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Year : 2012  |  Volume : 55  |  Issue : 1  |  Page : 72-74
Antifungal susceptibility testing of Candida tropicalis biofilms against fluconazole using calorimetric indicator resazurin

Department of Microbiology, Dr. A. L. Mudaliar Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, India

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Date of Web Publication11-Apr-2012


Background: C.tropicalis is an important cause of nosocomial infections particularly in immunocompromised patients. Infections caused by Candida spp. are often associated with biofilm formation on implanted medical devices or on epithelial cell surfaces. Phenotypic characteristics of sessile cells in biofilms are known to be different from those of their free-living, planktonic counterparts. Biofilm forming strains often show increased resistance to antimicrobial agents. Materials and Methods : We measured susceptibility to fluconazole of fifty C.tropicalis isolates from immunocompromised (29) and immunocompetent (21) patients by minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) assays. MBIC was done using the calorimetric indicator resazurin, to measure the metabolically active cells. Results : Biofilm forming cells showed increased resistance to fluconazole. Conclusion : The resazurin dye test was found to be a good method for determining MBIC.

Keywords: Biofilms, C. tropicalis, fluconazole, minimum biofilm inhibitory concentration, resazurin

How to cite this article:
Punithavathy P M, Nalina K, Menon T. Antifungal susceptibility testing of Candida tropicalis biofilms against fluconazole using calorimetric indicator resazurin. Indian J Pathol Microbiol 2012;55:72-4

How to cite this URL:
Punithavathy P M, Nalina K, Menon T. Antifungal susceptibility testing of Candida tropicalis biofilms against fluconazole using calorimetric indicator resazurin. Indian J Pathol Microbiol [serial online] 2012 [cited 2022 Sep 26];55:72-4. Available from:

   Introduction Top

Candida species are an important cause of infections, particularly in immunocompromised and critically ill patients. Although Candida albicans is the predominant etiological agent of candidiasis, other Candida species such as Candida tropicalis have emerged as important opportunistic pathogens in immunocompromised individuals. A wide range of biomaterials are used in clinical practice and they often represent a risk factor for the development of nosocomial Candida infections. [1] Biofilm formation occurs as a consequence of the device being colonized by the microorganisms. Biofilms are defined as communities of microorganisms that grow on an abiotic or biotic surface and are embedded in a matrix consisting of an extracellular polymeric substance.

The increased resistance to antimicrobial therapy is associated with the biofilm mode of growth and explains why biofilm-associated infections often results in treatment failure. [2] The therapeutic concentrations of antifungals are predicted by the results of antifungal susceptibility tests; however, occasionally, the antifungal susceptibility data do not correlate with the clinical outcome. One of the reasons for the lack of correlation is related to the biofilm-forming ability of Candida species, since standard antifungal tests done according to CLSI guidelines test only free planktonic cells.

It is therefore necessary to have a standard antimicrobial susceptibility testing protocol to test sessile cells. A variety of methods have been used for the quantification of biofilms such as direct microscopic enumeration, total viable plate counts, with use of metabolically active dyes, radiochemistry, and luminometry. [3],[4] A promising method for the evaluation of viability or activity of organisms in biofilms after the exposure to antifungals appears to be the use of calorimetric indicator resazurin, a component of Alamar blue. [5] Metabolically active cells reduce this resazurin to a pink product resorufin.

In this study, we use an easy, rapid, inexpensive conventional 96-well microtitre plates to perform an in vitro antifungal susceptibility testing method for C. tropicalis biofilms based on assessment of minimum biofilm inhibitory concentration (MBIC) of antifungal agent using redox indicator resazurin.

   Materials and Methods Top

Yeast Isolates

A total of 50 clinical isolates of C. tropicalis were used in the course of this study, of which 29 isolates (oral swabs (20), bronchial wash [4], urine [2], endotracheal secretion [1], sputum [1], and vaginal swab [1]) were from immunocompromised patients (HIV and cancer) and 21 isolates (urine (19), oral swab [1], and vaginal swab [1]) were from immunocompetent patients. A standard strain of C. tropicalis ATCC 13803 was also included. The strains were stored in glycerol stocks at -80°C until use. All the isolates were speciated using standard mycological procedures like color on CHROM agar, germ tube test, and sugar assimilation and fermentation tests.

Antifungal Susceptibility Testing

Minimum inhibitory concentration of fluconazole by broth microdilution method

Antifungal susceptibility testing was performed as per CLSI-M27-A2 recommendations. The stock solution of fluconazole was diluted with RPMI to achieve concentrations ranging from 0.25 to 256 μg/ml. Inoculum suspension was prepared from fresh cultures in sterile 0.85% saline matching 0.5 McFarland standards (1 × 10 6 to 5 × 10 6 CFU (Colony Forming Units)/ml).

The broth microdilution test was performed by using sterile, disposable, multiwell microdilution plates (96 -U shaped wells). 100 μl aliquots of serially diluted antifungals were dispensed into microtitre plate wells and 100 μl of test inoculum was added to each well. The plates were incubated at 35°C and read at 24 hours by visual inspection. Minimum inhibitory concentration (MIC) of ≤8 μg/ml was interpreted as sensitive, ≥64 μg/ml as resistant, and 16 to 32 μg/ml as susceptible dose dependent.

Minimum biofilm inhibitory concentration testing

The MBIC testing was performed by the technique using the calorimetric indicator resazurin, [6],[7] with minor modifications. Isolates were inoculated on fresh Sabouraud's dextrose agar (SDA) plates and incubated at 37°C for 24 to 48 hours. The yeast cultures from SDA were resuspended in 0.9% NaCl (sodium chloride) and opacity adjusted to 3 on the Mc Farland scale. The stock solution of resazurin dye was prepared by diluting the resazurin sodium salt in distilled water at 0.01% (w/v) which was filter sterilized and then added to RPMI medium (buffered to pH 7.0 using 165 mmol/L3 - N-morpholino) propanesulfonic acid) in a 1:10 ratio (0.001%).

Biofilms were formed on commercially available, presterilized, polystyrene, flat-bottom 96-well microtiter plates. Biofilms were formed by pipetting 90 μl of Sabouraud's dextrose broth supplemented with 8% glucose and 10 μl of standardized cell suspensions (prepared as above) into wells of the microtiter plate and incubating them for 48 hours at 37°C as described above. After biofilm formation, the medium was aspirated, and the planktonic cells were removed by thoroughly washing the biofilms three times in 0.9% sterile saline. Sterile paper towels were used to remove the residual saline from the microtiter plate, following which the antifungal agents were added.

Different concentrations of fluconazole (1 to 2 048 μg/ml) were made from stock solutions using RPMI medium with 0.001% resazurin dye. 100 μl of each dilution of the antifungal agent was aseptically added to the wells and incubated for a further 48 hours at 37°C to detect the viability of biofilms. A series of wells without the antifungal agent and uninoculated wells served as positive and negative controls, respectively. MBIC was determined as the lowest concentration of the antifungal agent maintaining the blue color of calorimetric medium.

   Results Top

The antifungal agent fluconazole showed decreased activity against C. tropicalis sessile cells, compared with their planktonic forms. Fifty isolates of C. tropicalis were tested for fluconazole susceptibility by broth microdilution method. MIC of 31 (62%) isolates were sensitive, eight (16%) were sensitive dose dependent and 11 (22%) isolates were resistant to fluconazole [Table 1]. The standard strain of C. tropicalis ATCC 13803 was sensitive (MIC, 0.5 μg/ml).
Table 1: MIC of fluconazole against C. tropicalis planktonic forms by broth micro dilution

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Among the 50 isolates tested by MBIC, all isolates were found to be resistant. Seven (14%) isolates had MBIC of 512 μg/ml and all of them were from immunocompetent patients, 28 (56%) isolates had MBIC of 1 024 μg/ml, and 12 (24%) isolates had MBIC of 2 048 μg/ml. Three (6%) isolates had MBIC of >2 048 μg/ml and all of them were from immunocompromised patients [Table 2]. Hence, MBIC values of sessile forms were higher than the MIC of planktonic forms. Metabolically active cells were seen even with high concentrations of the antifungal agent [Figure 1]. Antifungal susceptibility tests done in triplicates gave similar results.
Table 2: MBIC of fluconazole against C. tropicalis sessile cells

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Figure 1: Resazurin dye test for determining minimum biofi lm inhibitory concentration of fluconazole to C. tropicalis biofilm. Growth in the medium is indicated by change in color from blue to pink

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   Discussion Top

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, [8] slow growth rate, [9] and surface-induced expression of resistant genes. [10]

Resistance to fluconazole in Candida isolates may be either intrinsic or acquired. Immunocompromised patients often harbor strains which have acquired resistance due to continuous or intermittent exposure to the antifungal agent. Patients with recurrent disease, such as HIV patients with mucosal candidiasis, may carry only a single strain, but as CD4 counts decrease and frequency of infections increase, higher therapeutic doses of antifungal agents are used each time and the MIC gets progressively higher. The practice of administering azole antifungal agents to immunocompromised patients for prophylaxis is another reason for the high rate of resistance seen in this group of patients. [11]

Routine antifungal tests detect resistance in planktonic forms and standard methodologies that allow evaluation of antifungal agents against cells in biofilms are not frequently employed. Metabolically active cells in biofilms can be identified by disrupting the biofilm layer by means of sonication, followed by determination of CFU/ml. But this method appears to be laborious and inaccurate because the biofilm matrix cannot be completely disassociated. [12]

The viable cells in the biofilms after its exposure to antifungals can be conveniently measured by calorimetric methods which are easy to read because of clear-cut end points. The most commonly used method, e.g., reduction of tetrazolium to formazan, [13] is laborious and expensive; other disadvantages include toxicity due to the chemicals and requirement of addition of an electron-coupling reagent. [12]

Others researchers [14],[15] have also shown the usefulness of resazurin/alamar blue as a cell viability indicator in antifungal susceptibility testing. Our results corroborate previous observations [6],[13] indicating the increased resistance of Candida biofilm cells to antifungal agents.

The use of resazurin dye test in antifungal susceptibility testing of biofilm-forming cells has benefits like simplicity, low cost, lack of toxicity, and easy determination of end points.

   References Top

1.Crump JA, Collignon PJ. Intravascular catheter-associated infections. Eur J Clin Microbiol Infect Dis 2000;19:1-8.  Back to cited text no. 1
2.Baillie GS, Douglas LJ. Candida biofilms and their susceptibility to antifungal agents. Methods Enzymol 1999;310:644-56.  Back to cited text no. 2
3.Hawser S. Adhesion of different Candida spp. to plastic: XTT formazan determinations. J Med Vet Mycol 1996;34:407-10.  Back to cited text no. 3
4.Schierholz JM, Beuth J, König D, Nürnberger A, Pulverer G. Antimicrobial substances and effects on sessile bacteria. Zentralbl Bakteriol 1999;289:165-77.  Back to cited text no. 4
5.O'Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000;267:5421-6.  Back to cited text no. 5
6.Kuhn DM, George T, Chandra J, Mukherjee PK, Ghannoum MA. Antifungal susceptibility of Candida biofilms: Unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother 2002;46:1773-80.  Back to cited text no. 6
7.Bachmann SP, VandeWalle K, Ramage G, Patterson TF, Wickes BL, Graybill JR, et al. In vitro activity of caspofungin against Candida albicans biofilms. Antimicrob Agents Chemother 2002;46:3591-6.  Back to cited text no. 7
8.Samaranayake YH, Ye J, Yau JY, Cheung BP, Samaranayake LP. In vitro method to study antifungal perfusion in Candida biofilms. J Clin Microbiol2005;43:818-25.  Back to cited text no. 8
9.Douglas LJ. Candidabiofilms and their role in infection. Trends Microbiol 2003;11:30-6.  Back to cited text no. 9
10.Ramage G, Saville SP, Thomas DP, López-Ribot JL. Candida biofilms: An update. Eukaryot Cell 2005;4:633-8.  Back to cited text no. 10
11.Badiee P, Alborzi A, Shakiba E,Farshad S, Japoni A. Susceptibilityof Candida species isolated from immunocompromised patients to antifungal agents. East Mediterr Health J 2011;17:425-30.  Back to cited text no. 11
12.Pettit RK, Weber CA, Kean MJ, Hoffmann H, Pettit GR, Tan R, et al. MicroplateAlamar blue assay for Staphylococcus epidermidis biofilm susceptibilitytesting. Antimicrob Agents Chemother 2005;49:2612-7.  Back to cited text no. 12
13.Ramage G, Vande Walle K, Wickes BL, López-Ribot JL. Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 2001;45:2475-9.  Back to cited text no. 13
14.Pfaller MA, Barry AL. Evaluation of a novel calorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates. J Clin Microbiol 1994;32:1992-6.  Back to cited text no. 14
15.Tiballi RN, He X, Zarins LT, Revankar SG, Kauffman CA. Use of a Calorimetric system for yeast susceptibility testing. J Clin Microbiol 1995;33:915-7.  Back to cited text no. 15

Correspondence Address:
Thangam Menon
Department of Microbiology, Dr. A. L. Mudaliar Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.94861

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  [Figure 1]

  [Table 1], [Table 2]

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