Antifungal Susceptibility and Biofilm Production of Candida spp. Isolated from Clinical Samples

Objective The study aims to speciate clinical Candida isolates and detect their biofilm-forming ability and antifungal resistance. Methods All the Candida spp. isolated from different clinical samples like pus, urine, blood, and body fluid were included in the study. Biofilm production was tested by the microtiter plate method. Antifungal susceptibility was studied by the disk diffusion method. Patient's demographic details such as age, sex, and clinical information were collected. Presence of other risk factors such as diabetes mellitus, history of antibiotic use, and any urinary tract instrumentations was also recorded. Results Among 90 Candida species isolated, most predominant species was found to be C. albicans (45.5%) followed by C. tropicalis (28.88%), C. krusei (20%), C. glabrata (3.33%), and C. parapsilosis (2.22%). Candida spp. were isolated from urine (43%), BAL/sputum (18.88%), high vaginal swab (8.88%), suction tips (7.77%), blood and wound swabs (6.66%), pus (3.33%), bile aspirate (2.22%), and deep tissue (1.11%). A larger number of females were affected than males, and the age group of 51 to 60 years was more susceptible to candidiasis. A higher number of C. albicans isolates produced biofilm followed by C. parapsilosis, C. tropicalis, and C. krusei. However, C. glabrata showed no biofilm production in our study. All Candida isolates were 100% sensitive to amphotericin B. Voriconazole was the next effective drug with 81.11% susceptibility. 24.44% of strains were resistant to fluconazole. Conclusion Speciation of Candida isolates, detection of ability to form the biofilm, and monitoring of antifungal susceptibility testing are necessary for appropriate treatment.


Introduction
Recent advances in research technology have allowed researchers to study bacteria and fungi in their natural environment, and over 95% of bacteria existing in nature are in biofilms [1]. Candida species are found as normal flora in healthy individuals and are known to cause opportunistic infections with high rates of mortality, especially in immunocompromised individuals [2]. Candida spp. cause systemic diseases which are the fourth leading cause of nosocomial bloodstream infections in modern hospitals. e most challenging clinical problem is the increased rate of non-Candida albicans isolation and the rapidly growing resistance of Candida species [3].
Candida albicans is the most prevalent among Candida spp., which causes both superficial and systemic infections.
A majority of the diseases caused by Candida spp. are due to biofilm formation. Biofilms are the group of microorganisms that are embedded in an extracellular matrix (ECM), forming a complex three-dimensional architecture on biotic and abiotic surfaces [6]. Biofilm formation can occur on mucosal surfaces and plastic surfaces of indwelling devices. Biofilms are genetically resistant to antifungal agents including amphotericin B (AMB) and fluconazole (FLU). Biofilm formation varies depending on the Candida spp. [7]. Most frequently, pathogenic effects are caused by Candida albicans and to a lesser extent by other Candida spp. [8].
Candida shows resistance to azole due to general and long-term use of it [9]. Candidiasis can reoccur repeatedly. Some health-care providers prescribe antifungal drugs on a long-term basis, but this can lead to drug-resistant candidiasis that is more difficult to treat. erefore, early identification of Candida spp. and monitoring their antifungal susceptibility help in treatment. Since there are very few studies on biofilm formation and drug resistance reported from India, the present study is undertaken to isolate Candida species from various clinical specimens, detect biofilm formation, and to study their antifungal susceptibility pattern. e genus Candida was identified by colony morphology, Gram-staining, and other standard biochemical reactions [10]. All the media and chemicals used in the study were procured from Hi-Media Laboratories Pvt. Ltd., Mumbai, India.

Data Collection.
Patient's demographic details such as age, sex, and clinical information were collected. Presence of other risk factors such as diabetes mellitus, history of antibiotic use, and any urinary tract instrumentations was also recorded from the hospital information system.

Antifungal Susceptibility
Testing. Fungal susceptibility to routinely used drugs like amphotericin B (100 units), fluconazole (25 μg), and voriconazole (1 μg) was done by the disk diffusion method, using Mueller-Hinton agar supplemented with 0.5 mg/ml methylene blue. Agar plates were inoculated with a suspension of yeast cells whose turbidity was adjusted to 0.5 McFarland standards (10 6 CFU/ml) in a manner that is currently being used for testing antibacterial agents. Antifungal disks were placed on the inoculated plates and incubated at 27°C for 24-48 hours. e diameter of the zone of inhibition was measured. Results were interpreted as per CLSI guidelines [11,12].

Biofilm Production by Microtiter Plate Method (MTP).
Biofilm formation was performed on a sterile 96-well microtiter plate. A colony of each isolate was inoculated into tubes containing two ml of brain heart infusion broth (BHIB) and incubated at 37°C for 24 hours. All the broth cultures were diluted at a ratio of 1 : 20 using fresh BHIB, and 200 μl was placed into microtiter plates and incubated at 37°C for 24 hours. After the completion of incubation, microtiter plates were emptied, rinsed with distilled water three times, and then inverted to blot. Each well was then filled with 200 μl of 1% crystal violet and incubated for 15 mins. After incubation, the microplates were again rinsed three times with distilled water. en 200 μl of ethanol: acetone mixture (80 : 20 w/v) was added to each well and were read at 450 nm using an ELISA reader, and OD was recorded for each well. Sterile BHIB without microorganisms was used as the negative control. e cut-off value was determined by arithmetically averaging the OD of the wells containing sterile BHIB and by adding a standard deviation of +2. Samples with an OD higher than the cut-off value were considered positive, whereas those with the lower optical density than the cut-off were deemed to be negative [13].

Results
In the present study, 90 Candida spp. were isolated from different clinical samples, which included C. albicans (45.5%) followed by C. tropicalis (28.88%), C. krusei (20%), C. glabrata (3.33%), and C. parapsilosis (2.22%). ey were subjected to antifungal susceptibility testing and biofilm formation. Results are summarized as frequency tables, and percentages were worked out. Statistical analysis was performed by using Statistical Package SPSS V.16.0. Proportions and association were calculated by the descriptive study test and unpaired Student's t-test. Table 1 shows various Candida spp. isolated from clinical specimens. In the present study, 41 Candida spp. were obtained from males and 49 from females as shown in Table 2.
Clinical history was obtained from the medical records. Among them, 25 patients had diabetes mellitus, nine were catheterized, 21 had signs and symptoms of UTI, four were cancer patients, eight were pregnant women, 14 with chronic kidney disease, 17 with a respiratory infection, and one had heart disease.
Antifungal susceptibility testing of various Candida species showed that all isolates were susceptible to amphotericin B. e result of antifungal susceptibility of different Candida spp. is shown in Table 4.

Discussion
Candida spp. is found to be commensal, and interruption of standard host defense is required for them to act as pathogens. As there is an increase in the number of patients who are immunocompromised, aged, receiving antibacterial and aggressive cancer chemotherapy, or undergoing invasive surgical procedures and organ transplantation, Candidiasis has emerged as an alarming opportunistic disease [14].
In the present study, it is found that candidiasis can occur at all ages and both sexes. e youngest being a oneyear-old infant, while the oldest was 88 years. According to surveys conducted by Emeribe et al. [18] and Puri et al. [19] Candida infection was found the maximum in the age group of 21-40 years. In our study, maximum isolation of Candida spp was found in the age group of 51 to 60 years. Higher susceptibility of older adults to Candida infection could be due to lower immunity, aging, predisposing factors such as diabetes mellitus and malignancies. More number of females were affected than males with an incidence of 49 (54%) and 41 (45%), respectively, which is similar to the study done by earlier workers. e rate of isolation of Candida spp. in females was about 61.2%, while in males, it was only 38% [15]. In the present study, the distribution of Candida spp. among the males and females was found to be similar.
One of the most frequent predisposing factors in our study was diabetes mellitus which accounted for 28% of the total cases, followed by patients with symptomatic urinary tract infection (24%), pregnancy (8.8%), and catheterized chronic kidney disease (15.5%), respectively. However, Arora et al. demonstrated intravenous cannula (63%) followed by prolonged use of antibiotics (35%) and immunosuppression (23%) to be the most common risk factors in their study [20]. e risk factors may vary from geographical areas and may also be due to underlying conditions and food habits/immunity of patients studied.
Biofilms are universal, complex, interdependent communities of surface-associated microorganisms, enclosed in an exopolysaccharide matrix occurring on any surface, including medical devices [21]. e pathogenicity of Candida species is associated with its ability to form Biofilm and is an essential virulence determinant during candidiasis [14]. e present study showed biofilm production to be 57.14% among non-C. albicans spp. and 39.02% among C. albicans.
is result is nearly similar to those reported by Kumar et al. [22].
Among the non-Candida albicans spp. studied for biofilm production in the present study, 100% of C. parapsilosis showed biofilm formation, followed by C. tropicalis (61.53%) and C. krusei (55.55%). However, Kuhn et al. showed that C. albicans produces quantitatively more biofilms than other Candida species [23]. is difference could be due to the usage of different methods like silicone elastomer for biofilm production and tetrazolium for quantification. However, in the present study, the microtiter plate method with crystal violet stain was used. C. glabrata did not show biofilm production in our study. e chisquare test showed a nonsignificant variation in the number of biofilm producers and nonproducers.
It is crucial to monitor antifungal resistance among Candida species because it gives clues to emerging threats of new resistant strains that help in empirical treatment.
Among the 90 Candida isolates tested, all were susceptible to amphotericin B in our study which is similar to the results reported by Arora et al. [ [27]. Our resistance rates of fluconazole and voriconazole are in line with those of earlier studies. e possibility of reduced susceptibility to fluconazole and voriconazole may be due to widespread and long-term use of those antifungals among the study subjects.
ere is no statistical correlation between the biofilm formation and antifungal susceptibility (p > 0.05).

Conclusion
Candida infection depends on the underlying conditions of the patients. Antifungal resistance is emerging in our Candida isolates. However, there was no correlation between biofilm formation and antifungal resistance among our candidal strains.

Data Availability
e experimental data used to support the findings of this study are available from the corresponding author upon request.
Ethical Approval e study was approved by the Institutional Ethics Committee of Kasturba Medical College, Mangalore, Karnataka.

Conflicts of Interest
e authors declare that they have no conflicts of interest.