Biofilm Formation and Antifungal Susceptibility of Candida Isolates from Various Clinical Specimens

Background: Candida species are now recognized as major causative agents of hospitalacquired infection. Aims: To evaluate the species distribution, biofilm formation,and antifungal susceptibility (amphotericin B, ketoconazole, and fluconazole) of Candida isolates. Place and Duration of Study: This is a Six-months Cross sectional study conducted in Alansar hospital, Al-Madinah, Saudi Arabia. Methodology: One hundred and three isolates of Candida spp. were cultured on Sabouraud dextrose agar (SDA). Candida spp. were identified by four standard methods, CHROMagar candida, cornmeal agar, germ tube test and API 20C. Detection of Biofilm formation was done by microtitre plate and antifungal susceptibility testing was done by disc diffusion. Results: C. albicans was the most common species 61%, followed by C. tropicalis 25%, C. lusitanaie 5%, C. parapsilosis 4%, C. glabrata 4% and C. famata 1%. Biofilm formation was found to occur most frequently among non-albicans spp.(70%) than C. albicans (46%). All Research Article British Microbiology Research Journal, 3(4): 590-601, 2013 591 isolates were sensitive to amphotericin B and ketoconazole. Resistance to fluconazole was found in 22.5% of non-albicans spp. and 5% of C. albicans isolates. Conclusion: The present study proved that C. albicans is still the major isolate from urinary, vaginal and respiratory samples but non-albicans spp. predominate in the blood samples and from plastic devices. The non-albicans spp. were more biofilm producers compared to C. albicans and C. tropicalis showed the highest score of biofilm intensity (grade 4+). The species isolated are less susceptible to fluconazole.


INTRODUCTION
Candida spp. are the most common cause of fungal infections leading to a range of invasive life threatening to non-life-threatening diseases. Candida spp. has been recognized as the fourth commonest cause of nosocomial invasive infections [1]. Candidiasis has emerged as an alarming opportunistic disease as there is an increase in number of patients who are immune-compromised, aged, receiving prolonged antibacterial and aggressive cancer chemotherapy or undergoing invasive surgical procedures and organ transplantation. Among Candida spp., C. albicans is the most common frequent causes of infection, but non-albicans spp. Infections are increasing [2].
The important factors contributing to the virulence of candida is the formation of surfaceattached microbial communities known as "biofilms" [3]. Biofilms are attached to a surface and encased in a matrix of exopolymeric material. A typical laboratory fungal model of biofilm formation involves three operational steps: (a) adhesion, (b) biofilm growth, and (c) maturation [4]. Biofilm formation helps the organism to evade host defenses, exist as a persistent source of infection and develop resistance against antifungal agents. Candida spp. are frequently found in the normal microbial flora of humans, which facilitates their encounter through implanted biomaterials and host surfaces [5]. The resistance of biofilm forming candida spp. to antifungal agents represents a major challenge especially in the design of therapeutic and prophylactic strategies [6] .
Amphotericin B, a polyene fungicidal agent, has been standard treatment for candidal infections for decades, but the toxicity of the conventional form and the coast of its lipid forms limit its use. More recently, azole antifungal compounds, with lower toxicity and perfect efficacies, have emerged as the principal drugs used in treatment of candidal infection. Disk diffusion has served as simple, rapid, and cost-effective method for screening the susceptibility patterns of the yeasts [7].
The aim of this study was identification of Candida spp. isolated from various sources, detection of biofilm formation among isolates and in-vitro antifungal susceptibility of the isolates to amphotericin B, ketoconazole, and fluconazole.

Isolation and Identification of the Candida Isolates
A total of 103 non repetitive different clinical samples were collected from patients being treated in Al-ansar hospital, Al-Madinah, Saudi Arabia from August 2012 till February 2013. Isolates were from different sites of the body including respiratory tract (n= 41,40%), blood (n=14, 13%), urine (n=26, 25%), genital tract (n=10, 10%),plastic devices (n=8, 8%), and other sources (n=4,4%).all samples were cultured onto Sabouraud Dextrose Agar (SDA) (Himedia, Mumbai, India). Isolates were identified by four standard methods, CHROMagarcandida (CHROMagar company, BD), cornmeal agar, germ tube test (Table 1 and Fig. 1) and API 20C (Bio-Merieux, France). API 20C strip (20 cupules) were inoculated according to the manufacturer's instructions, incubated at 30ºC and observed at 48 and 72h. Reactions were read by comparing the strips to growth controls and identification was obtained by referring to the analytical profile index. C. albicans ATCC 10231 was used for quality controls [8].The collected Candida species strains were stored in 10% glycerinated water at −20ºC until biofilm was performed.

Antifungal Susceptibility Testing
The in vitro activity of antifungal agents (amphotericin B, ketoconazole, and fluconazole) was measured by disk diffusion method according to the procedure described in the clinical and laboratory standard institute [9]. document M44-A. The plates were incubated at 35°C, and inhibition zone diameters were measured after 24 and 48 h especially for C. glabrata. The interpretive criteria for the disk test were as follow: amphotericin B: dz ≥15mm, susceptible; 14≥dz≥10mm, susceptible dose dependent and dz≤9mm, resistant. Fluconazole: dz ≥19mm, susceptible; 15≤dz≤18mm, susceptible dose dependent and dz≤14mm, resistant. As for ketoconazole: dz≥20mm, susceptible; 10<dz<20mm, susceptible dose dependent and dz≤10mm, resistant [10].

Biofilm Formation
A total of 103 Candida spp. isolates were grown in Sabouraud Dextrose Broth (SDB) at 30ºC for 18 h. Centrifugation was done for 5 minute at 4000 rpm and washed twice with saline. The pellet was then re suspended in 5ml of sterile saline, and turbidity adjusted to 3 of the McFarland scale. To each well of the microtiter plates (flat bottom) 180µl of SDB with 8% glucose were added, then inoculated with 20µl of the above yeast cell suspension. Yeastfree medium controls were also included. The plates were then incubated for 48 h at 37ºC. After incubation, plate growth was assessed by measuring the absorbance at 490nm. The wells were washed thrice with sterile saline. A volume of 200µl of 1% crystal violet (Sigma, USA) was added to each well, and the plate was allowed to stand for 20 minutes at room temperature. The wells were subsequently washed thrice with sterile saline. A volume of 200µl of ethanol was added to each well and absorbance was measured at 630nm with a microtiter plate reader PEBIII (Dade Behring). The values above negative control were considered as biofilm producers [11].The percent transmittance (%t) was measured, the %t value for each test sample was subtracted from the %t value for the reagent blank to obtain a measure of the amount of light blocked when passing through the wells (%t bloc ). Biofilm production by each isolate was scored as either negative (%t bloc , < 5), 1+ (%t bloc , 5 to 20), 2+ (%t bloc , 20 to 35), 3+ (%t bloc ,35 to 50), or 4+ (%t bloc , >50) [12].

Distribution of
Biofilm formation in various clinical samples: The results of biofilm formation were also analyzed with respect to the site of infection, and revealed that the biofilm positivity was observed more with plastic devices 6/8 (75%) and bloodstream isolates (71%), urine samples were moderately biofilm producers 15/26 (58%) and the least isolates for biofilm production were from HVS 3/10 (30%) and respiratory tract 21/41 (51%) ( Table 4).
In vitro antifungal susceptibilities of various Candida spp. Antifungal susceptibility testing in our study showed that all isolates were susceptible to amphotericin B and ketoconazole.

Surgical departments
Medical departments

Other departments
Resistance to fluconazole was found in 22.5% of non-albicans spp. and 5% of C. albicans isolates with statistical significant difference between two groups (P= 0.01) ( Table 5).

DISCUSSION
Candida organisms are commensals; and to act as pathogens, interruption of normal host defenses is necessary. Candidiasis has emerged as an alarming opportunistic disease as there is an increase in number of patients who are immunocompromised, aged, receiving prolonged antibacterial and aggressive cancer chemotherapy or undergoing invasive surgical procedures and organ transplantation [2].
Our study indicated an increased prevalence of the Candida spp. In ICUs (57%) and surgical wards (22%). Similar results were reported by Sheevani et al, 2013. This can be explained on the basis of the fact that the use of invasive devices are common in these wards and that the immune status of the patients are also compromised to the maximum as compared to that of the patients of other wards [15 ].
The present study showed the distribution of Candida spp. In different clinical samples and the predominance was forC. albicans 61% (63/103) with statistical significant difference than non-albicans spp. (P=0.002). Similar to our results, C. albicans was the most common species isolated from different clinical sources [13 , 14]. On the other hand lower prevalence rates (39.5% and 25%) of C. albicans was reported by Mokaddas et al,2007 andChakrabarti et al, 1996, respectively. [16 , 17] Our study showed that C. albicans was the predominant species recovered from respiratory tract samples, Urine samples and HVS. These findings are consistent with those previously reported by other researchers [2 , 18 , 19] .In a recent studyC. albicanswas reported as the major agents of candiduria [20]. However other study showed that non-albicans spp., especially C. tropicalis and C. glabrata predominate in urine samples in many regions [21]. Emam et al, 2012 reported C. albicans as a predominant species isolated from pregnant women and non-pregnant women with vulvovaginitis [22].
Meanwhile our results showed that the predominant species isolated from the blood and plastic devices were non-albicans spp. and mainly C. tropicalis. These results are nearly similar to those reported by Hasana et al, 2009 [23].Most catheter-related septicemias are caused by microorganisms that invade the intracutaneous wound during catheter insertion or thereafter [12]. The proportion of such infection due to non-albicans spp. is persistently rising [24,25].
However other studies reported that the common isolate from the blood stream infection was C. albicans [26] and others reported C. parapsilosis rather than C. tropicalis as a predominant non albicans spp. isolates from blood stream [27] and reported that C. parapsilosis followed C. albicans as the most frequently isolate from candidemia clinical cases. Other studies on the epidemiology of candidaemia, have stressed the importance of the speciation of Candida, as it provides accurate information on the disease incidences and the trends in most of the infections [28]. Kothavade et al, (2010) reported that C. tropicalis as the most prevalent pathogenic yeast species of the non-albicans spp. [29] A biofilm is a community of microorganisms and their extra cellular polymers that are attached to a surface [30] . The ability to form biofilms is associated with the pathogenicity and as such should be considered as an important virulence determinant during candidiasis. Biofilms may help to maintain the role of fungi as commensal and pathogen, by evading host immune mechanisms, resisting antifungal treatment, and withstanding the competitive pressure from other organisms. Consequently, biofilm related infections are difficult to treat [31] .
Our study revealed that a statistically significant difference (P=0.02) regarding the biofilm formation was found among non-albicans spp. 28/40 (70%) than C. albicans 29/63 (46%). These results are nearly similar to those reported by many othrerreaserchers [32 , 33]. Mohandas and Ballal, 2011 concluded that the biofilm formation is more important for non-albicans spp. and C.albicans possess mechanisms other than biofilm formation to establish infections [2].
Our study showed that among the non-albicans spp., the biofilm positivity occurred most frequently among isolates of C. parapsilosis (100%), followed by C. tropicalis (73.1%) also C. tropicalis showed the highest score of biofilm intensity 12/18 (66.6%) was of grade (+4). C. tropicalis have also been recognized as strong slime producers in many other studies [2,1325 ]. However, Kuhn et al, (2002) showed that C. albicans produces quantitatively more biofilm than other Candida spp., but in that study the assessment of biofilm was based on quantitation and fluorescent microscopic examination proving that the biofilm formed by pathogenic C. albicans was a complex phenomenon composed of blastospore layer coved by a thick biphasic matrix, consisting of a dense extracellular component comprised of cell wall-like compounds and abundant hyphal elements composed of polysaccharide elements [34] .
Our data provide evidence that the majority of Candida spp. recovered from the plastic devices (75%) and bloodstream isolates (71%) have higher capacity to produce biofilm. Similar results were obtained by other studies [6,35]. Kuhn et al, (2002) reported that Invasive C. albicans isolates form more biofilm than noninvasive isolates [34] . Candida spp. are frequently found in the normal microbial flora of humans, which facilitates their encounter through implanted biomaterials and host surfaces [5]. The devices become colonized by Candida which forms biofilm, the detachment of which can result in candidemia. Indwelling catheters therefore, represent a major risk factor associated with nosocomial candida infections [32] . On the other hand the least biofilm producer isolates were observed in HVS 3/10 (30%) and respiratory tract 20/41 (48.7%). These findings were in concordance with studies conducted by Golia et al., 2011 [6]. It could readily be hypothesized that for certain body sites, colonization requires a particular phenotype with respect to biofilm formation. Such a biofilm phenotype might be genetically rather than environmentally governed [36].
In this study the resistance of all the isolated Candida spp. to fluconazole was 11%. The study by Nemati et al (2008), reported that the rate of resistance to fluconazole among Candida spp. ranged from null to the 15% [37]. Furthermore, our data on the fluconazole against C. albicans, revealed that 95% of tested strains were susceptible. This sensitivity rate is more or less comparable with those rates of 87.5% and 89.5% previously reported by Citak et al, (2005) and Badiee and Alborzi (2011), respectively [38,39]. In agreement with the study of Sabatelli et al. (2006) [40], most of the detected resistant strains belong to nonalbicans spp. (22.5%), emphasizing, its greatest potential to acquire resistance to fluconazole. Also, in agreement with the finding of Ng et al (2000) who reported, amphotericin B and ketoconazole susceptibility data and showed that all yeast isolates were susceptible. The resistance to antifungal agents such as fluconazole has been noted among non-albicans isolates. The possibility of increase in the percentage of the resistance to antifungal agents among non-albicans spp. Is due to widespread use of antifungal drugs, long-term use of suppressive azoles, and the use of short courses of antifungal drugs [10].

CONCLUSION
The present study proved that C. albicans is still the major isolate from urinary, vaginal and respiratory samples but non-albicans spp. predominate in the blood samples and from plastic devices. Biofilm positivity occurred most frequently in isolates of C. parasilosis followed by C. topicalis, C. lusitaniae, C. albicans, and C. glabrata. Non-albicans spp. commonly resistant than C. albicans to fluconazole agents.

ETHICAL APPROVAL
Ethical committee of Al-ansar hospital approved the study.