DRBC agar: a new tool for Candida dubliniensis identification ...

Folia Microbiol. 55 (2), 155–158 (2010)

http://www.biomed.cas.cz/mbu/folia/

DRBC Agar: a New Tool for Candida dubliniensis Identification L. ALVES SCHEID, D.A. NUNES MARIO, I.H. SOARES, É. SILVA LORETO, J.M. SANTURIO, S. HARTZ ALVES* Department of Microbiology and Parasitology. Universidade Federal de Santa Maria, Santa Maria (RS), 97010-033, Brazil Received 8 April 2009 Revised version 22 October 2009

ABSTRACT. Candida dubliniensis pathogenic species, which shares many phenotypic features with C. albicans, may be misidentified in the microbiology laboratory. The growth on DRBC agar at 25 °C was shown to be a new tool for differentiation between C. dubliniensis and C. albicans. All 27 isolates of C. dubliniensis showed in this medium rough colonies (peripheral hyphal fringes) and abundant chlamydospore production, while all 103 isolates of C. albicans showed smooth colonies without fringes or chlamydospores. DRBC agar allowed the differentiation of C. albicans from C. dubliniensis with 100 % sensitivity and specificity.

Abbreviations DRBC HIV FN

dichloran–Rose Bengal–chloramphenicol (medium) human immunodeficiency virus false negative (results)

FP TN TP

false positive true negative true positive

Candida dubliniensis was described as a new species of Candida in Ireland by Sullivan et al. (1995). Moreover, it was first associated with oropharyngeal candidiasis in AIDS patients (Sullivan et al. 1995; Coleman et al. 1997; Jabra-Rizk et al. 2001), and it has more recently been isolated from many other pathologies common in immunocompromised patients, including diabetic patients (Willis et al. 2000), cystic fibrosis patients (Peltroche et al. 2002) and cancer patients (Sebti et al. 2001). It has also been identified as a cause of systemic disease (Chan-Tack 2005). This species has been misidentified as Candida albicans because isolates of both species show similar phenotypic characteristics, such as the presence of a germ tube chlamydospore production and similar biochemical patterns (Sullivan et al. 1995; Loreto et al. 2008; Ells et al. 2009). These similarities restrict the development of epidemiological studies with C. dubliniensis. Currently, the reliable identification of C. dubliniensis requires molecular techniques (Neppelenbroek et al. 2006; Ells et al. 2009). Some studies have reported new alternative phenotypic methods to be routinely applied in medical microbiology laboratories, including morphological characterization on chromogenic and plant-derived media, growth at elevated temperatures and growth in the presence of sodium chloride (Ells et al. 2009). Here, we report the use of dichloran–Rose Bengal–chloramphenicol (DRBC) agar in the discrimination between C. albicans and C. dubliniensis based on hyphal fringe chlamydospore production. MATERIALS AND METHODS Strains. A total of 100 clinical isolates of C. albicans were obtained from the Laboratório de Pesquisas Micológicas – LAPEMI (Department of Microbiology; Universidade Federal de Santa Maria, Brazil) and were recovered from various clinical specimens, mostly respiratory tract and onychomycosis cases. C. dubliniensis clinical isolates (n = 26) were provided by the LAPEMI yeast collection, and all of them were recovered from oral cavities of HIV-infected patients. C. albicans ATCC 28367, C. albicans ATCC 44373, C. albicans ATCC 10231 and C. dubliniensis CBS 7987 were also included as reference strains. The definitive identification of all clinical isolates of C. dubliniensis had previously been confirmed by the random amplification of polymorphic DNA using the primers CDU (5´-GCG ATC CCC A-3´) (Sullivan et al. 1995) and B-14 (5´-GAT CAA GTC C-3´) (Bauer et al. 1993).

*Corresponding author; fax +55 21 3220 8906, [email protected] .

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Culture media. DRBC agar (g/L): glucose 10, peptone 5, agar 15, KH2PO4 10, MgSO4·7H2O 0.5, Rose Bengal 0.025, dichloran (2,6-dichloro-4-nitroaniline) 0.002, chloramphenicol 0.1 g (Atlas 2006). Niger seed agar was prepared according to Staib and Morschhäuser (1999). Phenotypic characterization. Each isolate was freshly subcultured on Sabouraud dextrose agar, and a single colony was separately streaked with a sterile wire loop on DRBC agar and Niger seed agar. All the plates were incubated for 1–2 d at 25 °C. The morphological characteristics were determined by direct observation of colonies at low-power (×10) and high-power (×40) magnifications. Data analysis. Niger seed agar and DRBC agar were evaluated in terms of the numbers of true positives (TP), true negatives (TN), false positives (FP) and false negatives (FN). The presence of hyphal fringes and chlamydospores was considered a true positive value, while the absence of them was considered a true negative value for C. dubliniensis identification. On the other hand, the absence of hyphal fringes and chlamydospores were considered a true positive value for the C. albicans identification. Sensitivity was calculated as TP/(TP + FN) and specificity as TN/(TN + FP). RESULTS AND DISCUSSION The observation of the growth of Candida spp. isolates on the Niger seed agar used as a control and on DRBC agar showed that all 27 (100 %) C. dubliniensis isolates grew on both media as rough colonies with hyphal fringes on their peripheral margins, appeared white- or cream-colored on Niger agar and pinkcolored on DRBC agar and formed chlamydospores after 1–2 d of incubation at 25 °C. Although chlamydospores were present in 100 % of C. dubliniensis isolates, their numbers were variable. In contrast, all 103 C. albicans isolates (100 %) showed smooth white or cream colonies on Niger seed agar and smooth pink colonies on DRBC agar; hyphal fringes and chlamydospores were absent (Fig. 1). Both Niger seed agar and DRBC agar showed 100 % sensitivity and specificity in differentiating C. albicans from C. dubliniensis for the analyzed strains.

Fig. 1. Morphological characteristics under optical microscope of the colonies of C. albicans (A – 10× and C – 40×) and C. dubliniensis (B – 10× and D – 40×) on DRBC agar.

Specific media have been proposed to discriminate C. dubliniensis from C. albicans, such as Niger seed agar (Staib and Morschhäuser 1999), sunflower agar (Al Mosaid et al. 2003) and others (Khan et al. 2004; Loreto et al. 2006, 2008; Ells et al. 2009) (Table I). Simple and cheap methods for presumptive dif-

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ferentiation of these two species described but it is well established that C. dubliniensis isolates present large phenotypic variability, which hinders the standardization of the identification techniques (Sullivan and Coleman 1998); therefore the search for new and more elaborated methods is advantageous and should be encouraged. Table I. Phenotypic differences between C. albicans and C. dubliniensis Characteristic Chlamydospores Color on chromogenic media Chromagar Candida Candida ID2 Candi Select 4

Candida albicans

Candida dubliniensis

+

+++

(light) green cobalt blue pink

dark green turquoise blue dark green

smooth colonies without fringes or chlamydospores

hyphal fringe and chlamydospore production

Growth Elevated temperature (45 °C) Sabouraud with NaCl 6.5 % Tween 80 opacity

+ + halo around their colonies

– – absence of opacity

Assimilation D-Xylose Methyl--D-glucoside

+ +

– –

Plant-derived media Niger seed (Staib) agar Sunflower (Pal’s) agar Sesame and linseed agar Rosemary agar

DRBC agar – isolation, cultivation and enumeration of yeasts and moulds. In its primary purpose, the Rose Bengal suppresses the growth of bacteria and restricts the size of mould colonies. This restriction aids in the isolation of slow-growing fungi by preventing them from being overgrown by more rapidlygrowing species. Rose Bengal is taken up by yeast and mould colonies, thereby facilitating their recognition and enumeration. Likewise, the antifungal agent dichloran is added to reduce colony diameters of spreading fungi (King et al. 1979; Atlas 2006). The initial observation of abundant chlamydospores produced by C. dubliniensis (in pairs or triplets on the ends of short-branched hyphae) in contrast with the single chlamydospore on the tip of hyphae showed in C. albicans isolates on cornmeal-Tween or rice-Tween agar is not reliable because C. dubliniensis and C. albicans showed no consistent pattern of chlamydospore formation (Kirkpatrick et al. 1998; Kurzai et al. 2000; Ellepola et al. 2003; Álvarez et al. 2009). On CHROMagar, the contrast between light-green and dark-green Candida colonies can be subtle. Moreover, the observations that C. albicans isolates also produced dark green colonies (Kurzai et al. 2000; Mähnß et al. 2005) and that C. dubliniensis might lose the characteristic dark green color after subculturing and storage (Pincus et al. 1999) limit the use of this medium in primary isolation. The growth of C. albicans at 45 °C and the absence of growth of C. dubliniensis at this temperature is a characteristic that should be analyzed in association with other phenotypic tests because some C. albicans isolates also failed to grow at 45 °C (Kirkpatrick et al. 1998; Gales et al. 1999; Kurzai et al. 2000). The hyphal fringe and chlamydospore production observed in C. dubliniensis on plant-derived media represents one of the best options for routine laboratory use. However, one study with a larger collection of C. dubliniensis showed that 14.6 % of the isolates were no longer able to produce chlamydospores on Niger seed agar (Al Mosaid et al. 2001). In opacity tests on Tween 80 agar, some C. albicans isolates failed to show the characteristic halos around the colonies (Dolapci et al. 2004). Growth in hypertonic Sabouraud broth requires a longer incubation time, and some C. albicans isolates do growth well in this high salt concentration (Alves et al. 2002). All these tests are simple and cheap but suffer from low standardization due to variations in the composition of the media, and the fact that these media have only a single use in the laboratory has prevented them from being widely used. In contrast, the main advantage of DRBC agar is the possibility of acquiring it commercially; in addition, it is quick to use and has a well-known application in the microbiology laboratory.

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