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Year : 2015  |  Volume : 33  |  Issue : 5  |  Page : 112--114

Biofilm as a virulence marker in Candida species in Nosocomial blood stream infection and its correlation with antifungal resistance

M Bhatt1, G Sarangi1, BP Paty1, D Mohapatra1, N Chayani1, A Mahapatra2, P Das3, D Sahoo1,  
1 Department of Microbiology, Shri Ramachandra Bhanj Medical College and Hospital, Cuttack, India
2 Department of Microbiology, All India Institute of Medical Sciences, Bhubaneswar, Odisha, India
3 Department of Microbiology, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India

Correspondence Address:
M Bhatt
Department of Microbiology, Shri Ramachandra Bhanj Medical College and Hospital, Cuttack


Nosocomial blood stream infections (BSI) due to fungi especially Candida is increasing steadily. A two year prospective study was conducted in the S.C.B. Medical College with an aim to evaluate the species distribution, antifungal susceptibility and biofilm formation of Candida spp. isolated from nosocomial BSIs. 34 Candida spp. were isolated from 359 blood cultures. Antifungal susceptibility was performed by microbroth dilution technique and both visual and spectrophotometric method were used for biofilm detection. C. tropicalis was the common spp. isolated followed by C. parapsilosis and others. Most (92%) of the isolates were susceptible to Amphoterecin-B and highest resistance was observed against Flucytosine (37%) and Fluconazole(35%). Biofilm production and antifungal resistance was observed more in nonalbicans Candida spp.

How to cite this article:
Bhatt M, Sarangi G, Paty B P, Mohapatra D, Chayani N, Mahapatra A, Das P, Sahoo D. Biofilm as a virulence marker in Candida species in Nosocomial blood stream infection and its correlation with antifungal resistance.Indian J Med Microbiol 2015;33:112-114

How to cite this URL:
Bhatt M, Sarangi G, Paty B P, Mohapatra D, Chayani N, Mahapatra A, Das P, Sahoo D. Biofilm as a virulence marker in Candida species in Nosocomial blood stream infection and its correlation with antifungal resistance. Indian J Med Microbiol [serial online] 2015 [cited 2019 Dec 12 ];33:112-114
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Nosocomial (Blood stream infections) BSIs are growing problems in tertiary care hospitals throughout the world. Bacteria have been the traditional etiology of nosocomial BSIs, but with increasing use of broad spectrum antibiotics, central venous catheters (CVC) and indwelling devices, fungi have superseded bacteria. [1] Among fungi, Candida spp. is the commonest and is the fourth most common cause of BSI in hospitalised patients. [2] Candida spp. possesses a number of virulence factors including biofilm production. Biofilms are the structured microbial communities that are attached and encased in a matrix of exopolymeric material. [3] Candida spp. has the ability to form extensive biofilm on catheters and other prosthetic devices which contributes to its prevalence as an etiologic agent of intravascular nosocomial infections. Central venous catheters are considered as the most common risk factor for the development of candidemia. [4] As a virulence factor biofilm not only enable Candida spp. for haematogenous dissemination but also renders to high level antimicrobial resistance. [5] Hence early detection of biofilm production can be an useful aid for clinical decisions. Various methods like Tissue culture plate, Congo red, Tube and Microtitre plate are available for biofilm detection. [6],[7],[8]

The present study was aimed to isolate and identify Candida spp. from suspected nosocomial BSIs, followed by biofilm detection and antifungal susceptibility testing.

 Materials and Methods

A 2 years study (Aug 2009-Aug 2011) was conducted in the Department of Microbiology, S.C.B Medical College, Cuttack. Study population included patients admitted to different intensive care units (ICUs), dialysis units, and oncology wards having nosocomial BSI. Nosocomial BSI was diagnosed in patients who developed infection after being hospitalised for >48 hours which was neither present nor in the incubation during hospitalisation. Total 359 blood samples were collected from patients who have had no antifungal exposure during hospitalisation. Samples were inoculated into brain heart infusion biphasic medium (BHIBPM) and incubated aerobically at 37° C. Candida spp. were isolated after appearance of colony on BHIBPM and identified by colony morphology, Gram stain, Germtube test, Sugar assimilation, Sugar fermentation and Chlamydospore formation on cornmeal agar. Biofilm production was determined and correlated by using the methods proposed by Branchini et al., and Ruzicka et al.[7],[8] Inoculum of Candida isolates were prepared in Sabouraud's dextrose broth supplemented with 8% glucose and turbidity adjusted to Mc farland's 0.5. Ten millilitre broth in tubes and 20 μlt in microtitre wells were incubated at 37° C for 24 hours and 48 hours, respectively, after which the broth was aspirated out and stained with safranin. In tubes, biofilm production was observed visually by two separate observers and correlated. Biofilm formation was considered positive when a visible film lined the walls and bottoms of the tubes but ring formation at the liquid interface was not considered. The absorbance of the microtitre wells were measured at wavelength 630 nm by Micro ELISA auto reader. A blank well with reagent was also measured for absorbance [% of transmittance (%t) blank]. The %t was substracted from %t blank to obtain the amount of light blocked. Biofilm production was scored as negative, 1+, 2+, 3+ and 4+ when %t blank = <5, 5-20, 20-35 and >50, respectively. Experiment was performed in triplicate and the data was then averaged.

All the isolates were subjected to antifungal susceptibility testing by microbroth dilution technique using amphotericin B, voriconazole, fluconazole, itraconazole and 5-flucytocine as per CLSI M-27-A2,2002. [9] Inoculum was prepared using Roswell Park Memorial Institute 1,640 medium with glutamin phenol red and 0.2% glucose at PH 7 and standardised to 0.5 Mc Farland. Antifungal powders were procured from commercial sources and stock solutions were made with DMSO and tested in serial dilution for each antifungal with different concentrations. Tests were interpreted after 24 hours incubation at 37° c by visual method with reading mirror and compared with controls (C. albicans ATCC 90028 and C. parapsilosis ATCC 95142).


A total of 34 (9.4%) Candida spp. were isolated among which 13 (39%) were C. tropicalis, 7 (20%) C parapsilosis, 5 (14.7%) each of C. kruseiand C. Albicans and 2 (5.4%) were C. glabrata. Two isolates could not be identified up to species and were not subjected to biofilm production [Table 1]. Among the 32 isolates subjected for biofilm production, 22 (64.7%) were positive along with the standard strains. No major discrepancies were observed between visual and spectrophotometric reading. Strong biofilm production (3+, 2+) was seen in 13 strains and weak (1+) in 9 strains. C. parapsilosis and C. tropicalis were found to be strong biofilm producers whereas C. Albicans and C. krusei were identified as weak producers. Antifungal susceptibility showed 5-Flucytocine to be the most resistant drug followed by fluconazole. Amphotericin B was found as the most susceptible antifungal agent [Figure 1], [Figure 2].{Figure 1}{Figure 2}{Table 1}


Blood stream infections by fungi were prevalent since long but the incidence of nosocomial candidemia has increased dramatically over the last 2 decades. [10] Approximately, 200 Candida spp are known, but only 10% are recognised as human pathogens and C. albicans was the single most common species causing nosocomial BSI so far. Recent reports suggest a shift towards non-albicans Candida spp. like C. tropicalis and C. parasilosis. [11],[12] The prevalence of nosocomial candidemia in the present study was found to be 9.4% which correlates approximately with that of Xess et al., (6%) from All India Institutes of Medical Sciences (AIIMS) and Sahni et al., (6.9%) from Maulana Azad Medical College (MAMC) New Delhi. [1],[10] Risk factors for candidaemia are increasingly associated with ICU patients as being subjected to invasive procedures, IV catheterisation and hyperelimentation. [10],[13] In the present study 100% were administered broad spectrum antibiotics, 89% had indwelling venous catheters and 65% were mechanically ventilated during their stay in the hospital. Nonalbicans Candida spp. predominated (85.3%) in our study which is well correlated with studies from different parts of India. Xess et al., had reported 86.4% of non-albicans Candida spp from BSI at a tertiary care hospital in North India. [10] The predominant nonalbicans Candida spp. in this study is C. tropicalis 13 (39%) followed by C. parapsilosis 7 (20%) and others. The predominance of C. tropicalis among non-albicans Candida have also been reported by Shiv Prakash et al., (35.6%) and Adhikary et al. (39.7%). [11],[14] The exact cause for this could not be explained however increased use of fluconazole could be the major contributing factor. [12] Moreover, C. parapsilosis is found commonly on skin surface and has better adherence to materials like acrylic, glucose and parenteral nutrition solutions which is considered very similar to the concentration in milieuinterior. [4] Hence the detection of C. parapsilosis in BSI is an indication of exogenous infection. [12] Biofilm production was detected in 64.7% of the isolates in this study among which non-albicans Candida spp. (C. tropicalis and C. parapsilosis) were strong producers. Another study had similar findings of non-albicans Candida as strong biofilm producer but their overall rate of biofilm production was higher in comparison to ours. [8] The antifungal susceptibility results showed highest resistance to 5 Flucytosine and Fluconazole (37% and 35%, respectively) although Voriconazole, Itraconazole and Amp-B showed good efficacy. Western studies reported Candida spp. to be remarkably susceptible to Polyenes, Flucytosine, Azoles and Echinocandins. [15] But an Indian study had reported very high resistance toVoriconazole (56%) and Fluconazole (36%) although Amp-B susceptibility was as high (92%) as ours. [14] We observed higher degree of resistance to Fluconazole in biofilm producers as compared to non-producers as reported by several authors. [5] Possible resistance mechanism involves restricted penetration of drug through biofilm matrix, phenotypic changes resulting from a decreased growth and nutritional limitation, expression of resistance genes induced by contact with a surface and persistence of a small number of cells.


To conclude, non-albicans candidaemia is on rise and biofilm production is responsible for increasing antifungal resistance. The changing epidemiology of candidaemia, therefore, highlights the need for close monitoring on the distribution, susceptibility and biofilm production of Candida spp. in order to optimise therapy and outcome.


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