Characterization and Biofilm Detection among Clinically Important Candida Species

 

Varshan R¹, Dr. Gopinath P^2*

²Department of Microbiology, Saveetha Dental College and Hospital, Chennai, Tamilnadu, India.

 

ABSTRACT:

Background: Candida is the fourth most common cause of blood stream infection in hospitalized patients. Candidiasis is associated with intravenous lines and prosthetic lines are problematic, as they can act as substrates for biofilm production, the propensity of forming this is more in immuno suppressed patients. Materials and methods: A total of 20 clinical isolates of Candida spp were obtained from different clinical conditions of different immuno compromised patients and were processed for its characterization followed by assessing the biofilm production. Results: Of 20 isolates, 40% were C. albicans, 35% were C. tropicalis, 15% were C. krusei and each of 5% were C. kefyr and C. parapsilosis respectively. 5% of isolates were strong biofilm formers whereas, 65% were moderate biofilm producers in our Candida isolates. Conclusion: The present study reveals there was an increase in degree of isolation of non-albicans Candida spp from immuno compromised individuals. These isolates were moderately producing biofilm. Hence, prompt identification and detection of biofilm are essential in clinical setup where more number of Candida have been encountered.

 

 

INTRODUCTION:

Mycotic infections are the major cause of mortality and morbidity in immuno compromised patients. Of all fungal agents candid species play a predominant role in causing infections.^[1] Candida species are the flora of an induviduals mucosal oral cavity, GIT and vagina^[2] and are associated with different clinical conditions from oral thrush to blood stream infection.^[3]

 

These agents may cause symtematic infections in such patients due to their great adaptability to different host environments. Candida is a heterogenous group of organism, comprises of more than 17 different Candida species which are known to cause infections to humans.

 

However more than 90% of deep infections are caused by C.albicans, C.glabrata, C.parapsilosis, C.tropicalis and C.krusei.^[4] Candida specuea possesses a number of virulent determinants, biofilm production is one among them. Biofilms are the structural microbial communities attached and encased in a matrix of eco polymeric substances.^[5]

 

C. species has the ability to exhibit biofilm on catheters and other prosthetic devices which contributes to its prevalance in various nosocomial infections as an etiological agent.^[6]

 

MATERIALS AND METHODS:

CANDIDA ISOLATES

A total of 20 non repetitive clinical isolates of Candida species were collected from different samples of immunocompromised individuals attending Saveetha Medical college, Thandalam. They were characterized by carbohydrate fermentation and assimilation tests and confirmed. ^[7] Isolates were preserved in semisolid Sabouraud chloramphenicol semi solid stock and stored at 4˚C until further use.

 

CHARACTERIZATION OF CANDIDA SPECIES

Candida species were further characterized by using Hichrom agar (Himedia, Mumbai).

 

Preparation of Hichrom agar

CHROMagar Candida (HiMedia, Mumbai) was prepared following manufacturer's instructions. About 21.02 gram of HiChrome Candida differentiation agar base (modified) was suspended in 500 ml of distilled water. It was heated to boiling gently to dissolve the medium completely. Then it was allowed to cool to 50^oC and rehydrated (one vial) contents of Hichrome Candida selective supplement was added under aseptic precautions. It was mixed well and poured into petridishes. Isolates were identified on Hichrome agar based upon the characteristic color of the colony by subculturing from Sabouraud's chloramphenicol agar plates and the Candida Hichrome plates were incubated at 37^oC for 24- 48 hours.^[8] Based on colour produced by the isolates speciation have been made.

 

 

BIOFILM PRODUCTION BY TISSUE CULTURE PLATE METHOD (TCP)

Overnight grown cultures of Candida spp from agar plates were inoculated in 0.5% of glucose and incubated at 37˚C overnight. Individual wells of sterile polystyrene 96 well flat bottom tissue culture plates were filled with 200µL of culture suspensions from afore mentioned broths. Uninoculated broth served as negative control. The plates were incubated at 37˚C for overnight. After incubation , content of each well was gently discarded by tapping the plates downwards . The wells were washed three times with 200µL of PBS (pH 7.2) +in order to remove planktonic bacteria. Biofilms are formed by adherent sessile isolates in plates were fixed with 2% sodium acetate and stained with 0.1% W/V crystal violet. Excess stain was removed by washing the wells with distilled water and plates were kept for drying at an inverted position. Optical density of stained adherent bacteria was determined with an ELISA reader (BioRad) at wavelength 570nm. These OD values were taken as index of bacteria adhering the surface and formed biofilm. Experiment was carried out in triplicate and their mean was taken for the analysis.^[9]

 

 

 

The below mentioned interpretation charge was applied to categorize the ability of Candida to form biofilm

MEAN OD VALUES	ADHERENCE	BIOFILM FORMATION
<0.120	Non	Non / weak
.120-0.240	Moderately	Moderate
>0.240	Strong	High

 

RESULTS:

Characterization of Candida species by Hichrom Candida agar

A total of 20 clinical isolates of Candida spp were seeded on to Hichrom Candida agar and results were tabulated based on pigment production.

 

 

Figure 1: Pie Chart Showing Distribution of Candida Species

 

 

FIGURE 2: Representative picture showing Candida species on Hichrom Candida agar

 

Sample wise distribution of Candida species

Of the 20 clinical isolates of Candida spp, 6/20 (30%) were from oral thrush, 5/20 (25%) from urine, 3/20 (15%) from sputum, 3/20 (15%) from vaginal swab, 2/20 (10%) from ear swab and one (5%) from wound swab.

 

Figure 3: Pie chart showing the sample wise distribution of Candida species

 

 

Figure 4: Representative picture showing biofilm production of Candida species by TCP method.

 

BIOFILM PRODUCTION

All isolates were subjected for biofilm assay by tissue culture plate method. Of 20 Candida isolates 1/20 (5%) were found to be strong biofilm producers, 13/20 (65%) and 6/20 (30%) isolates were shown to be moderate and non-biofilm formers of Candida respectively.

 

BIOFILM CATEGORY	Candida Isolates (n=20) (%)
Non / Weak	6 (30)
Moderate	13 (65)
Strong	1 (5)

 

 

DISCUSSION:

In recent years, there has been a remarkable increase in the use of indwelling devices in hospitals. Infection of prosthetic devices with biofilm-forming microorganisms is a frequent problem. Candida species are important nosocomial pathogens. Modern medical procedures including implantation of various kinds of devices contribute to the risk factors for developing candidiasis. Sessile forms of microbes tend to be more resistant to antifungal agents than their planktonic forms. The mechanisms by which these forms exhibit increased resistance are not fully understood, but is believed to be due to complex mechanisms such as reduced diffusion of antimicrobial agents through biofilm layer, ^[10] slow growth rate, ^[11] and surface-induced expression of resistant genes^.[12]

 

Study conducted by Hassan et al. in 2011 from Pakistan shown that 64.7% of Candida spp were strong biofilm producers and 36.3% were weak formers of biofilm.^[13] Regional data from India also showed that out of 152 isolates tested, the number of biofilm producers identified by TCP method was 53.9 %, and non-biofilm producers were 46%.^[9] We have performed the TCP method by addition of 1% glucose in trypticase soy broth. Addition of sugar helps in biofilm formation.^[14] This was also reported by studies conducted by Mathur et al.^[9]

 

CONCLUSION:

From our study we have observed a marked increase in the non-albicans species of Candida isolated from immuno compromised individuals. However, Hichrom Candida agar is useful in preliminary identification of Candida spp by pigment production, there were a possibility in misidentifying the Candida spp producing similar pigments. As Candida spp are known to produce catheter related biofilm in ICU setup, but we have seen only quite less number of isolates were found to be biofilm formers. Thus, we conclude that it is very essential that assessing the biofilm forming ability of emerging Candida spp in hospital set up would throw more light on proper management and patient’s outcome. 

 

ACKNOWLEDGEMENT:

We thank Dr. Jeyalakshmi, Professor and Head of Microbiology Department for kindly providing the Candida isolates to carry out our research work successfully.

 

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Accepted on 25.06.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(9):1375-1378.