ANTIFUNGAL SUSCEPTIBILITY OF BIOFILM PRODUCING Candida albicans ISOLATED FROM ORAL CAVITY OF DIABETIC AND NON-DIABETIC POPULATION OF DHARAN, NEPAL

Background: Candida are almost universal on normal adult skin and C. albicans is part of the normal flora of the mucous membranes of the respiratory, gastrointestinal, and female genital tracts. It is acknowledged that diabetic patients are more susceptible to infections caused by Candida albicans due to increased blood glucose and inability of immune system in eradicating the fungus. Studies suggest that Gutkha consumers are also at high risk of oral Candida carriage . Materials and methodology The participants were provided 10 ml of Normal saline and were asked to oral rinse for 1 minute. Oral rinse was collected in a sterile screw capped container and was transported in cold chain to microbiology laboratory. The oral rinse sample was inoculated onto the Sabouraud dextrose agar with Chloramphenicol and was incubated at 37°C for 3-4 days. The Colony forming Unit of candida was compared among diabetic and healthy controls. The candida albicans were identified by Germ tube formation. The Candida albicans isolates were subjected to Biofilm assay, Antifungal susceptibility Test, Haemolysin assay, Haemolysis degree and phospholipase assay. Result: This study reported 31.5% prevalence of oral Candida.The Candida carriage in CFU of diabetic population was statistically significant (p<0.05). The maximum isolates were found to be Biofilm producers. There was significant association between Gutkha consumers with oral Candida carriage. The study suggests that there is higher colonization of Candida in diabetic populations than in healthy population. The result also concludes that frequency of Candida in Oral cavity of Gutkha consumers was also higher (p<0.05). All isolated strains of Candida albicans were tested for antifungal susceptibility testing and 76.19% were found to be Resistant to Fluconazole and 50% were found to be resistant to Amphotericin B. There was statistical significance in Biofilm formation and fluconazole Drug resistance.


Background
Candida albicans is known to be normal flora of human body harboring skin, mucosal cavity oral, vaginal mucosa and known to colonize 60% of healthy population [1]. Candida albicans is dimorphic fungi isolated most commonly from Biofilms of medical devises and human tissue [2]. Studies have suggested that Candida albicans are associated with number of opportunistic infections in immunocompromised patients like in HIV patients, cancer patients and diabetic patients etc. [3].
In diabetes mellitus type-II there has been known greater incidence of oral candidiasis which could be associated with immune dysfunction caused by high glucose concentration in blood, tissue and saliva [4]. Individuals with diabetes were found to have higher Candida carriage rate than the non-diabetics, presumably due to increased Candida growth with high glucose levels in saliva, blood and neutrophil dysfunction [5].
Candida has known to be opportunistic pathogen under immunosuppression and tobacco chewing conditions [6,7]. It is suggested that individuals chewing Gutkha are susceptible to oral Candida infections than non-chewers [8] .The greater prevalence and colonization of Candida in Gutkha consumers could be due to the presence of nicotine and hydrocarbons such as polycyclic aromatic hydrocarbons as a nutritional source for Oral yeast facilitating its growth [7.8]. Chewing Tobacco that includes Gutkha, Betel quid (BQ) which is common habit in South Asian nations like in India, Pakistan, Bangladesh, Sri lanka and Nepal [9]. Candida albicans pathogenesis is explained by its host defense mechanism, adherence, and production of tissue degrading hydrolytic enzymes like protease, phospholipase and haemolysin and role in biofilm production on host tissue and in medical devices [10]. Biofilm production is associated with the role of fungi to evade host immune function and overcome adhesions to host cell molecules to resist antifungal therapy making the treatment of infection more complicated [11].
Candida albicans exhibits hemolytic activity when grown on glucose-enriched blood agar [12]. In humans, most of the iron is located intracellular as ferritin or as heme-containing compounds. It is concluded that C. albicans expresses a hemolytic factor which allows it to acquire iron from host erythrocytes [13]. 4

Materials And Methods
Research design: The cross sectional lab based study was carried out in microbiology laboratory of Central campus of Technology, Hattisar from June 2018 to November 2018. During the study 200 oral rinse samples were analyzed. All the work concerning this research was carried out in microbiology laboratory in Central Campus of Technology, TU, Hattisar, Dharan. The different oral rinse samples analyzed were from different potential risk population (50-barbers, 50-municipal waste workers, 50diabetic and 50-healthy individuals).Ethical approval was obtained from Nepal Health Research Council, Kathmandu. The research participants were informed about the procedure of sample collection and the informed consent was obtained in written form. All the participants without oral lesions were included in the study. The Healthy population was selected by Nutritionist on basis of physical fitness, good oral hygiene whereas Diabetic population was selected on basis of blood glucose level obtained from their medical history. All the socio-demographic information like brushing habit, Gutkha chewing e.t.c was obtained from research participants through Schedule.

Inclusion criteria
Group I patients: In the inclusion criteria, 50-participant having DM without any other oral lesions with following criteria were included in the study. Participants who have not received antibiotic and corticosteroid therapy before 4 weeks were included in study.
Group II (Control group): 50-People who did not have DM or any other systemic illness, who were nutritionally fit, without any clinical signs of diseases, without any clinical medication were included as Healthy control.
Group III: It included 50-Barber population across the city.
Group IV: It included 50-Municipal waste worker of Dharan Sub metropolitan city.

Exclusion criteria
The people who never met above criteria were included under exclusion criteria Sample collection 5 Ten ml of sterile saline were allowed to be rinsed for 1 minute and inoculated in a broader capped sterile container. The oral rinse sample for study were collected from the sample population and transported to microbiology laboratory maintaining cold chain .All the collected samples were labeled with participant's identification number. In case of delay, the sample was usually stored at 4°C in the refrigerator.

Processing of oral rinse in SDA
An aliquot of 50 microliter of oral rinse sample were inoculated in SDA (HiMedia, Mumbai, India) with chloramphenicol (0.05 g/l) and incubated at 37°C for 24-48 hours. Pure culture was identified by colony characteristics, grams staining. The number of colony was counted by Colony Counter and expressed as CFU/ml [14,15].

Assessment of Candida colonization by Quantitative Enumeration
Number of colonies formed after incubation were counted by colony counter counted and multiplied with a factor of 20 to get the colonies in 1 ml of a subject's sample [15].

Identification of Candida albicans
Germ tube and Chlamydospore formation was evaluated as described by Beheshti et al (1975) [16].
Germ tube test: The pure isolated colony of candida albicans was dispensed in 0.5 ml of serum and incubated at 37°C for 2 hours. After incubation the aliquote was taken in a clean slide and was observed under microscope for the formation of Germ-tube.
Chlamydospore formation test: The pure isolated colony of Candida that could form Chlamydospore in Corn Agar was identified as Candida albicans.

Hemolysin Assay
Haemolysin activity was evaluated according to Luo et al 2001 [13]. Hemolysin production by Candida albicans was performed by inoculation overnight culture of yeast on Sugar-enriched sheep blood agar as described by Manns et al (1994) [13]. The Blood base agar media was prepared by adding 5-7 ml of fresh blood to Saboured Glucose Agar with 3% glucose. 10 µL of the yeast inoculum was placed at the centre of the plates. The plates were incubated at 37ºC in 5% CO 2 for 48 hours. Hemolytic Index (Hz value) which represents the Hemolysin production is the ratio calculated by dividing the total diameter of the colony plus the translucent halo by the diameter of the colony.

Screening Candida albicans for production of phospholipase
The phospholipase test was done according to Samranayakae et al (1984) [17]. Egg yolk agar media was inoculated by 2 µL of the inoculum and allowed to dry at room temperature. The plates will be incubated at 37ºC for 3-4 days. z will be measured by dividing the diameter of the colony by the sum of diameter of the colony and the zone.

Screening impact of diabetes in hemolytic activity
The hemolytic activities were tested on Saboured dextrose Broth liquid media (SDB) containing 7% defibrinated human blood according to Malcok et al (2009) [12]. One was supplemented with 3% glucose and the other without glucose. Candida albicans culture was inoculated and incubated for 48 hrs. The hemolysis in the media was detected spectrophotometrically by measuring the amount of released hemoglobin and compared with a standard hemolysate which was prepared prior to testing.
The degree of hemolysis (percentage value) by an individual strain was calculated according to the following formula below: (Absorbance of supernatant media at 540 nm/Absorbance of standard hemolysate at 540 nm X 100).

Microtitre plate method
The quantification of biofilm was performed according to Christensen et al (1985) [18]. In this method, 5 ml of overnight culture of Candida albicans was prepared. Then 100 microliter of diluted culture was inoculated in a sterile 96-well polystyrene well plates well containing TSB with glucose. The plate was 7 incubated at 37ºC for 24 hours for biofilm production. The unbound cell was discarded and washes several times. 125 μl of 0.1 % crystal-violet solution was added and left for 10-15 mins incubation. Optical density cut-off value (ODc) = average OD of negative control + 3x standard deviation (SD) of negative control.

Tube method
A qualitative assessment of biofilm formation was done as described by Christensen et al (1985) [18].
The TSB glu (10 mL) was inoculated with a loop full of Candida albicans from overnight culture plates and incubated for 24 hours at 37°C. The tubes was decanted and washed with PBS (pH 7.2) and dried.
Then the tubes were stained by 0.1% crystal violet. Stain was removed by deionized water. Tubes were then dried in inverted position for biofilm formation. Biofilm formation was considered positive when a visible film lined the wall and bottom of the tube. Ring formation at the liquid interface was not considered biofilm formation. An experiment was repeated for three times.

Congo red Agar Method (CRA)
The Candida albicans culture was streaked on surface of Congo Red Agar and incubated at 37ºC for 24-48 hours (Freeman et al 1989) [20]. Black coloured colonies with dry crystalline consistency interpreted as positive biofilm producing strains. Red coloured colonies-interpreted as negative for biofilm production.

Antifungal Susceptibility Testing
All Candida albicans isolated from samples were subjected to in-vitro antifungal susceptibility test by Kirby-Bauer disc diffusion method as recommended by CLSI 2010 [21]. In this study the antifungal

Statistical Analysis
The data was analyzed using statistical Package for Social Sciences (SPSS) version 16.0. Mann Whitney U test was carried out to calculate CFU counts, Chi Square (χ 2 ) were used for statistical analyses. The p value of equal or less than 0.05 was used for statistical significance.

Analysis of Candida Carriage between Diabetic cases and Healthy controls
Candida carriage expressed in CFU/ml was comparatively studies in both Case and control groups 9 using Mann Whitney U test. The result of statistical analysis of candida carriage is shown in Table 1 (Supplementary Files).

Prevalence of Candida in Population
The overall prevalence of Candida species was 31.5 % whereas the prevalence of Candida albicans only was 21 %. The result of prevalence of Candida yeast in population is shown in Figure 3.

Comparative study of Candida albicans isolated from different populations
Candida albicans were isolated from Diabetic population 29 followed by Barbers

Biofilm assay by microtitre plate method
The 96-well microtitre plate method for biofilm formation was carried out. The overall biofilm producers were 69.04 % and 35.96 % were non biofilm producer. The strong biofilm producer was 2 (4.76 %), moderate biofilm producer was 27 (64.28%) and Weak/none biofilm producer were 13 (30.96%). Biofilm assay by microtitre plate method is shown in Table 6 (Supplementary Files).
The Biofilm formation with Antifungal Drug Resistance is shown in Figure 5.

Sensitivity and Specificity of Biofilm Screening Methods
The microtitre plate method was found to be most efficient standard method for studying biofilm formation as compared to tube method and Congo red agar method. The parameters like sensitivity, specificity, negative predictive value, positive predictive value and accuracy were calculated and are shown in albicans in oral cavity of denture wearing males (74%) than in female (23%) with diabetic mellitus Type II disease. Loster et al (2016) reported that the infection free individual were greater in male than in female and statistically significant relationship between intensity of yeast and gender [23].
In our study the maximum frequency of organism was found in between age group of 40-50 years.
The association of Candida in in different age groups was found to be statically insignificant (p>0.05). Lockhart et al (1999) found the increase in oral candida carriage was reported by with age possibly due to diminished natural defenses [24]. In our study the prevalence of yeast in elderly age group was found to be least possibly due to least sample population from old age group. There was no significant association between Age and prevalence of Candida albicans.  [12].The mean hemolytic degree of Candida albicans in human blood with Glucose (SDBwG) was 58.68% and in human blood without glucose (SDBwoG) was 48.74%. We reported that albicans exhibiting higher haemolysin activity and 92.3% exhibiting phospholipase activity [30]. In our study all strains of C. albicans exhibited haemolysin activity and only 68% C. albicans exhibited phospholipase activity. In our study there was significant difference in Hemolysin activity among Diabetic and control groups (p=0.049).
Candida has known to be opportunistic pathogen under immunosuppression and tobacco chewing conditions [6,7]. It is suggested that individuals chewing Gutkha are susceptible to oral Candida infections than non-chewers [8]. Chewing Tobacco that includes Gutkha, Betel quid (BQ) which is 13 common habit in South Asian nations like in India, Pakistan, Bangladesh, Sri lanka and Nepal [31].
Betel quid is mixture of areca-nut, lime enveloped in piper betel leaf whereas Gutkha is found in Sachet [32]. The possible Explanation for greater Candida colonization in Gutkha consumers could be due to the presence of nicotine and hydrocarbons such as polycyclic aromatic hydrocarbons acting as nutrient for Oral yeast facilitating its growth [7.8] [34]. Even in our study The Microtitre method was considered efficient method for quantitative screening of biofilm in comparison to TM and CRA methods.
In our study 4.7% of isolated Candida albicans were strong biofilm producers, 64.2% were moderate and 30.9% were weak or non-biofilm producer. In overall the biofilm forming Candida albicans were 69% and non-biofilm producers were 30.9%. In our study the maximum frequency of Candida albicans were biofilm producers and even maximum biofilm producing Candida albicans were resistant to antifungal drugs. This association may explain the ability of biofilm production with drug resistance in the organism exhibiting its strong virulence factor and making failure of antifungal drugs. Mohammadi et al (2016) reported that biofilm formation and overgrowth of Candida is found more in diabetic patients [35]. Similar to it even in our study the antifungal resistance pattern of Candida albicans showed highest resistance to fluconazole whereas Amphotericin B also showed good sensitivity.  [36]. Similar findings were observed even in our study.
In Nepal, improper use of antibiotics drugs without doctor's prescription, haphazard selling and buying of antibiotic from local pharmacy and unnecessary use of drugs have been responsible for drug resistance to microorganisms. This uncontrolled use of drug has led to antifungal drug resistance in strains of microorganisms. Indiscriminate use of drugs in our community is a major cause for emergence of drug resistance. The proper availability of microbiological investigation and information would be necessary in search of appropriate antimicrobial agents to fight with drug resistance. Since for many years Fluconazole has been used for treatment of oral candidiasis but the emergence of drug resistance renders the search of alternative antimicrobial therapy.
The good oral hygiene and the reduced prevalence of Candida carriage was statistically significant, p<0.05. In our study the total Candida carriage among healthy population was only 26 %. Majima et al (2014) found 18.3% oral Candida carriage among dental students and suggested that good oral hygiene responsible for elimination of Candida [22]. Therefore with overall analysis in potential risk groups and in Healthy population, the oral Candida colonization shows a significant association with oral hygiene and serum glucose.
Higher prevalence of Candida in diabetic population and in poor oral condition alarms the oral health warnings. The absence of Candida in oral cavity may be due to maintenances of good oral health.
Thus, oral carriage of Candida serves for health issues as oral candidiasis requires initial colonization by Candida.   Hemolysis Degree exhibited by isolated Candida albicans in SDB with and without Glucose