Rapid differentiation of Candida albicans from other Candida species using its unique germ tube formation at 39 C

Transcription

1 Yeast Yeast 2002; 19: Published online in Wiley InterScience ( DOI: /yea.891 Research Article Rapid differentiation of Candida albicans from other Candida species using its unique germ tube formation at 39 C Donghwa Kim, 1 Woon-Seob Shin, 2 Kyoung-Ho Lee, 1 Kyunghoon Kim, 3 Joo Young Park 1 and Choon-Myung Koh 1 * 1 Department of Microbiology, Yonsei University Wonju College of Medicine, Wonju, Korea 2 Department of Microbiology, Kwandong University College of Medicine, Kangnung, Korea 3 Division of Life Sciences, College of Natural Sciences, Kangwon National University, Chunchon, Korea *Correspondence to: Choon-Myung Koh, Department of Microbiology, Yonsei University Wonju College of Medicine, 162 Ilsan-Dong, Wonju, Kangwon-Do, , S. Korea. kohcm@wonju.yonsei.ac.kr Received: 12 December 2001 Accepted: 30 April 2002 Abstract In this study, we found that no Candida species other than C. albicans is able to form germ tubes at 39 C in serum-free YEPD (1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) dextrose) media, which makes it easy to identify C. albicans from other Candida species. When cultivated in rabbit serum for at least 2 h at 37 C, more than 60% of C. albicans cells generated germ tubes. In YEPD, however, germ tubes began to appear from C. albicans cells within 30 min at 39 C, and more than 60% of C. albicans cells formed the germ tubes after 1 h at 39 C. Standard Candida strains (ATCC, CBS), three C. albicans and two C. dubliniensis strains were cultured in serum at 37 C for 2 h and in YEPD at 39 C for 1 h. All of the three C. albicans formed germ tubes at 39 C. The two C. dubliniensis strains formed germ tubes in serum at 37 C, but grew as a yeast form in YEPD at 39 C. All of the clinically isolated C. albicans strains in our laboratory formed germ tubes in YEPD at 39 Cfor1h, and none of the clinically isolated Candida species other than C. albicans generated germ tubes in YEPD at 39 C. Thus, the unique germ tube formation of C. albicans induced by high temperature (39 C) in YEPD could be applied to a protocol for the rapid and convenient identification of C. albicans in clinical laboratories. Copyright 2002 John Wiley & Sons, Ltd. Keywords: identification; Candida albicans; temperature; germ tube Introduction Rapid identification of Candida isolates to the species level in the clinical laboratory has become more important because the incidence of candidiasis continues to rise in proportion to a growing number of patients at risk for infection by Candida albicans (Elie et al., 1998). C. albicans is the most frequently isolated fungal pathogen in humans. As the primary agent of mucocutaneous and disseminated candidiasis, the opportunistic pathogen C. albicans causes a significant morbidity and mortality, especially in immunocompromised and/or postoperative patients (Greenfield, 1992; Hurley et al., 1987; Jarvis, 1995; Pfaller, 1995). In immunocompromised patients the clinical appearance of the C. albicans infection is often very complex and identification of the organism is difficult. Therefore, speedy diagnosis and management of candidiasis are crucial for these patients. C. albicans exhibits the ability to grow as either a yeast or a mycelial form in response to different environmental factors. In vivo, the mycelial forms found in infected tissues may be important as a virulence factor in the adherence of the organisms to host epithelial tissues (Cutler, 1991; Odds, 1988). In vitro, a germ tube can be induced from the yeast cell and developed into the mycelial form. This morphological transition can be induced by changes in a variety of environmental factors, Copyright 2002 John Wiley & Sons, Ltd.

2 958 D. Kim et al. including ambient ph (Pollack, 1987), nutritional status (Barlow et al., 1974; Bruatto et al., 1993; Casanova et al., 1997; Mattia et al., 1982) and temperature (Odds, 1985). However, the mechanism whereby these factors induce germ tube formation in C. albicans is virtually unknown. For the identification of Candida isolates, conventional methods, including germ tube and chlamydospore formation and sugar assimilation tests, have been used (Bikandi et al., 1998). To perform the germ tube formation test, yeast isolates are incubated in serum at 37 C for 2 3 h (Lee et al., 1999). In this study, we found that no Candida species other than C. albicans is able to form germ tubes at 39 C in serum-free YEPD medium, which makes it easy to identify C. albicans from other Candida species. Identification of C. albicans, based upon the ability to form germ tubes at 39 C in serum-free media, yielded more reliable results than those by conventional methods, such as the serum-induced germ tube test, chlamydospore test and colony colour test on CHROMagar Candida media (Kirkpatrick et al., 1998). Materials and methods Organisms and culture Standard strains of C. albicans ATCC 10231, C. albicans ATCC (serotype A), C. albicans ATCC (serotype B) were obtained from the National Institute of Health, Seoul, Korea. C. dubliniensis CD36 and C. dubliniensis CM2 were kindly provided by David A. Stevens (Santa Clara Valley Medical Center, San Jose, CA). All the yeast cells were precultured in YEPD broth composed of 1% (w/v) yeast extract (Difco), 2% (w/v) peptone (Difco) and 2% (w/v) dextrose at 37 C for 18 h. Precultured yeast cells were washed twice with 10 ml sterile PBS [50 mm sodium phosphate buffer with 0.9% (w/v) sodium chloride, ph 7.2]. Concentrations of the yeast suspensions were measured using a haemocytometer and adjusted by adding PBS to the suspensions. Carbohydrate assimilation Carbohydrate assimilation test was performed as follows (Rousselle et al., 1994). Suspended yeast cells in PBS equivalent to McFarland Standard No. 0.5 were spread on a yeast nitrogen base agar plate (Difco) with no carbohydrate. Paper disks soaked in 1% (w/v) solutions of various carbohydrates were placed on the plate. The results were read after incubating the plate at 25 C for 2 3 days. Chlamydospore formation Suspended yeast cells were deeply streaked with a loop on a cornmeal agar plate (Difco) containing 1% Tween 80 and covered with glass slips. The plate was incubated under the condition of a higher humidity at 25 C for 3 days. Chlamydospores were observed using a light microscope. Germ tube formation Washed C. albicans cells (10 9 cells/ml) were diluted to cells/ml with rabbit serum or YEPD liquid, and incubated at 37 Cor39 C for 1 2 h. The germ tube was observed with a light microscope. In order to investigate the germ tube structure, the elongated daughter cells from the round mother cell without constriction at their origin were referred to as germ tubes, and constricted hyphae at the round mother cell were referred to as pseudohyphae. Growth on CHROMagar Candida medium The cells were either pipetted onto a CHROMagar Candida plate (CHROMagar, Paris, France) as 10 µl drops of the cell suspension containing yeast cells/ml or streaked out to single colonies from the suspension. Cultures on the plate medium were incubated at 37 C and examined after 2 days. Preparation of DNA and PCR Genomic DNAs were isolated from clinical isolates of Candida species using a simplified phenol/chloroform extraction method (Muller et al., 1998). The cells were grown overnight at 37 Cin 2 ml YEPD medium, harvested, and resuspended in 400 µl lysis buffer (2% Triton X-100, 1% SDS, 100 mm NaCl, 1 mm EDTA, 10 mm Tris HCl, ph 8.0). To this suspension, 400 µl phenol saturated with 10 ml Tris HCl, ph 8.0, and 0.2 g acidwashed glass bead ( µm in diameter) were added and vortexed heavily for 50 s. The mixture was centrifuged for 1 min, and the aqueous

3 Rapid differentiation of C. albicans 959 phase was mixed with 800 µl ethanol. The precipitated DNA was harvested, washed with 70% (v/v) ethanol, and resuspended in 100 µl water. For PCR reactions, 1 µl DNA solution was used as template DNA. A pair of primers, CAL5 (5 - TGTTGCTCTCTCGGGGGCGGCCG-3 ) and NL- 5CAL (5 -AGATCATTATGCCAACATCCTAGG- TTAAA-3 ), complementary to a segment of the gene encoding C. albicans 26S rrna were used for PCR reactions (Mannarelli and Kurtzman, 1998). The reactions were performed in a DNA thermal cycler with an initial denaturation step (3 min at 94 C) followed by 35 cycles of denaturation for 20 s at 94 C, annealing for 40 s at 66 C, and extension for 20 s at 72 C. After a final incubation for 4 min at 72 C, the PCR products of 175 bp were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Results Effect of temperature on germ tube formation of C. albicans The ability of C. albicans to form germ tubes in serum or YEPD medium was investigated at different temperatures of C (Figure 1). More than 60% of C. albicans cells generated germ tubes in serum at 35, 37 and 39 C. Although germ tubes Germ tube formation (%) 100 YEPD Serum Temperature ( C) Figure 1. Effect of temperature on the germ tube formation of C. albicans. With the initial cell concentration of cells/ml, C. albicans ATCC was cultured in serum (white bar) or YEPD medium (black bar) at C for 2 h were generated maximally in serum at 37 C, virtually no germ tube was formed from YEPD-grown C. albicans at 30, 35, and 37 C. Surprisingly, however, we found that germ tubes are formed really well from C. albicans cells grown in YEPD at 39 C, and the germ tubes were true germ tubes without constrictions at their origins. It took at least 2 h for C. albicans cells to generate the germ tubes sufficiently in serum. In YEPD, however, germ tubes began to appear from C. albicans cells within 30 min at 39 C, and more than 60% of C. albicans cells formed germ tubes after an incubation time of 1hat39 C. Identification by high temperature-induced germ tube The standard Candida strains were cultured in serum at 37 C for 2 h and in YEPD at 39 Cfor 1 h (Table 1). All of the three C. albicans formed germ tubes both in serum at 37 C and in YEPD at 39 C. In case of the two C. dubliniensis strains, they formed germ tubes in serum at 37 C, but grew as a yeast form or a short chain form in YEPD at 39 C (Figure 2). Identification by chlamydospore formation The standard Candida strains were streaked on a cornmeal agar plate and incubated for 3 days at 25 C (data not shown). The C. dubliniensis strains formed abundant chlamydospores, frequently arranged in triplets or clusters of contiguous pairs on short branching pseudomycelia. The C. albicans strains produced single terminal chlamydospores but other strains produced no chlamydospores. Table 1. Microscopic morphology of Candida spp. Strains YEPD at 39 C Morphology Serum at 37 C C. albicans ATCC Germ tube Germ tube C. albicans ATCC Germ tube Germ tube serotype A C. albicans ATCC Germ tube Germ tube serotype B C. dubliniensis CD36 Yeast form Germ tube C. dubliniensis CM2 Yeast form Germ tube With the initial cell concentration of cells/ml, each strain was incubated in serum (37 C, 2 h) or in YEPD (39 C, 1 h).

4 960 D. Kim et al. Table 2. Sensitivity of identifying methods for clinical isolates of Candida species Germ tube induction Serum, 37 C YEPD, 39 C Chlamydospore PCR CHROMagar C. albicans 103/ / / / /105 Others 28 /93 0/93 1/93 0/93 0/93 For the germ tube induction, with the initial cell concentration of cells/ml, each yeast culture was incubated in serum for 2 h at 37 C and in YEPD for 2 h at 39 C. Germ tube of other Candida was pseudohyphae. Identification of clinical isolates of Candida spp. We had previously identified 105 C. albicans strains among hundreds of clinical Candida isolates using the carbohydrate assimilation test. Clinical isolates of 198 Candida spp., including the 105 C. albicans strains, were tested by a series of identification methods, such as germ tube formation in serum or YEPD, chlamydospore formation, DNA amplification by PCR, and colony colour on CHROMagar. All of the 105 C. albicans strains formed germ tubes in YEPD at 39 C (Table 2), and appeared light green on CHROMagar plates (data not shown). A DNA fragment of 175 bp specific to the 26S rrna gene of C. albicans was amplified from genomic DNAs of all the 105 strains (Table 2). Two out of the 105 C. albicans failed to form germ tubes in serum at 37 C, and four of the 105 C. albicans formed no chlamydospore (Table 2). However, none of the 93 Candida isolates other than C. albicans generated germ tubes in YEPD at 39 C, and appeared light green on CHROMagar. The 175 bp DNA was not amplified from the 93 Candida, either. One out of the 93 formed chlamydospores, and 28 out of the 93 formed pseudohyphae (not true germ tubes) in serum. Discussion Although C. albicans cells reproduce normally by budding, giving rise to the formation of yeast cells, they frequently generate germ tubes under unfavourable conditions. The production of germ tubes results in conversion to a filamentous growth phase or a mycelial form. Moreover, the formation of pseudohyphae occurs by polarized cell division when yeast cells grown by budding have elongated without detaching from adjacent cells. Under certain non-optimal growing conditions, C. albicans can undergo the formation of chlamydospores, which are round spores with thick cell walls. These morphological transitions often represent a response of the fungus to changing environmental conditions and may permit the fungus to adapt to different biological niches (Chaffin et al., 1998). For more convenient identification of Candida spp., commercial methods, including germ tube and chlamydospore formation as well as sugar assimilation tests, have been developed (Odds, 1988; Rousselle, 1994). Recently, molecular methods including PCR have also been studied (Elie et al., 1998; Mannarelli and Kurtzman, 1998; Shin et al., 1997; Weissman et al., 1995). However, the observation of germ tube production in serum as a method for the presumptive identification of C. albicans has been widely used for many years, and has provided quite reliable results. Nevertheless, since some other Candida spp., e.g. C. dubliniensis, C. tropicalis and C. parapsilosis, can generate germ tubes or pseudohyphae in serum and their sugar assimilation patterns are similar to that of C. albicans, it is often difficult to discern C. albicans from other Candida spp. (Figure 2). We found that only C. albicans generates germ tubes at 39 C in serum-free YEPD medium (Figure 2) and that commercial media (Sabouraud, Czapek, etc.) had no effect on germ Figure 2. Photographs of germ tubes of Candida spp. induced by high temperature (left) or rabbit serum (right). At an initial cell concentration of cells/ml, each Candida was cultured in YEPD at 39 C for 1 h or in rabbit serum at 37 C for 2 h. Magnification 400. (A) C. albicans ATCC 10231; (B) C. albicans ATCC 36801; (C) C. albicans ATCC 36802; (D) C. dubliniensis CD36; (E) C. dubliniensis CM2. Arrows represent germ tube

5 Rapid differentiation of C. albicans 961 Germ tube by 39 C Germ tube by serum

6 962 D. Kim et al. tube formation at 39 C. Although all of the 105 C. albicans strains isolated in our laboratory generated germ tubes at 39 C in YEPD, none of the other 93 Candida isolates in our laboratory generated germ tubes at 39 C in YEPD (Table 2). These results clearly provide us a more sensitive and convenient method to identify C. albicans than those using the ability to form germ tubes in serum media (Figure 2), to form chlamydospores (data not shown), and to produce coloured colonies on a CHROMagar Candida plate (data not shown). Identification of Candida albicans by germ tube formation at 39 C is clearly more rapid and cheaper than that by PCR (Mannarelli and Kurtzman, 1998). In conclusion, the unique germ tube formation of C. albicans induced by high temperature (39 C) in YEPD could be applied to a protocol for the rapid and convenient identification of C. albicans in clinical laboratories. References Barlow AJ, Aldersley T, Chattaway FW Factors present in serum and seminal plasma which promote germ-tube formation and mycelial growth of Candida albicans. J Gen Microbiol 82: Bikandi J, San Millan R, Moragues MD et al Rapid identification of Candida dubliniensis by indirect immunofluorescence based on differential localization of antigens on C. dubliniensis blastospores and Candida albicans germ tubes. J Clin Microbiol 36: Bruatto M, Gremmi M, Nardacchione A, Amerio M Effect of glucose starvation on germ-tube production by Candida albicans. Mycopathologia 123: Casanova M, Cervera AM, Gozalbo D, Martinez JP Hemin induces germ tube formation in Candida albicans. Infect Immun 65: Chaffin WJ, Lopez-Ribot JL, Casanova M, Gozalbo D, Martinez JP Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol Rev 62: Cutler JE Putative virulence factors of Candida albicans. Ann Rev Microbiol 45: Elie CM, Lott TJ, Reiss E, Morrison CJ Rapid identification of Candida species with species-specific DNA probes. J Clin Microbiol 36: Greenfield RA Host defence system in interactions with Candida. J Med Vet Mycol 30: Hurley RJ, de Louvois J, Mulhall A Yeasts as human and animal pathogens. In The Yeasts, 2nd edn, Rose AH, Harrison JS (eds). London: Academic Press; Jarvis WR Epidemiology of nosocomial fungal infections with emphasis on Candida species. Clin Infect Dis 20: Kirkpatrick WR, Revankar SG, McAtee RK et al Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus-infected patients in North America by primary CHROMagar Candida screening and susceptibility testing of isolates. J Clin Microbiol 36: Lee KH, Shin WS, Kim D, Koh CM The presumptive identification of Candida albicans with germ tube induced by high temperature. Yon Med J 40: Mannarelli BM, Kurtzman CP Rapid identification of Candida albicans and human pathogenic yeasts by using short oligonucleotides in a PCR. J Clin Microbiol 36: Mattia E, Carruba G, Angiolella L, Cassone A Induction of germ tube formation by N -acetyl-d-glucosamine in Candida albicans: uptake of inducer and germinative response. J Bacteriol 152: Muller FM, Werner KE, Kasai M, Francesconi A, Chanock SJ, Walsh TJ Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption. J Clin Microbiol 36: Odds FC Morphogenesis in Candida albicans. Crit Rev Microbiol 12: Odds FC Candida and Candidosis. A Review and a Bibliography, 2nd edn. WB Saunders: London. Pfaller MA Epidemiology of fungal infections. J Hosp Infect 30(suppl): Pollack JH, Hashimoto T The role of glucose in the ph regulation of germ-tube formation in Candida albicans. JGen Microbiol 133: Rousselle P, Freydiere AM, Couillerot PJ, de Montclos H, Gille Y Rapid identification of Candida albicans by using albicans ID and Fluoroplat agar plates. J Clin Microbiol 32: Shin JH, Nolte FS, Morrison CJ Rapid identification of Candida species in blood cultures by a clinically useful PCR method. J Clin Microbiol 35: Weissman Z, Berdicevsky I, Cavari B Molecular identification of Candida albicans. J Med Vet Mycol 33: