Antonie van Leeuwenhoek 44 (1978) 15-24

15

G e r m - t u b e f o r m a t i o n b y a t y p i c a l strains o f Candida albicans FAYE F. OGLETREE, A. T. ABDELAL AND D. G. AHEARN

Department of Biology, Georgia State University, Atlanta, Georgia 30303, U.S.A.

OGLETREE,F. F., ABDELAL,A. T. and AHEARN,D. G. 1978. Germ-tube formation by atypical strains of Candida albicans. Antonie van Leeuwenhoek 44: 15-24. Atypical isolates of Candida albicans which failed to produce germ tubes in routine diagnostic procedures were examined for their ability to produce germ tubes in various media. Bovine serum was more effective than defined media for induction of germ tubes in the majority of isolates. A few strains formed appreciable germ tubes only in bovine serum with added thioglycollate or cysteine. One strain did not produce germ tubes in any medium. Germ-tube maturation appeared to be dependent upon mitochondrial RNA polymerase activity. The failure by an isolate to produce germ tubes, particularly in tests without strictly controlled conditions, does not preclude the possibility that the organism is C. albicans.

INTRODUCTION The germ-tube test is currently accepted as a reliable method for the identification of C. albicans (Robin) Berkhout (Taschdjian, Burchall and Kozinn, 1960; MacKenzie, 1962; Ahearn, Jannach and Roth, 1966; Joshi and Gavin, 1974). Media for the induction of germ tubes include human and animal sera (Taschdjian et al., 1960; MacKenzie, 1962; Ahearn et al., 1966; Dolan and Ihrke, 1971 ; Joshi et al., 1973a), egg albumin (Buckley and van Uden, 1963), various peptone media (Joshi, Gavin and Bremmer, 1973b; Joshi and Gavin, 1974), tissue culture medium 199 (Landau et al., 1964), and media containing rice extracts, oxgall and Tween 80 (Beheshti, Smith and Krause, 1975). When compared with conventional procedures, presumptive identifications based solely on germ-tube formation have been reported to be 95-100 percent accurate (Stenderup and Thomsen, 1964; Aheam et al., 1966; Joshi and Gavin, 1974). The importance of temperature, inoculum density, media composition and strain variation in germ-tube formation has been documented for isolates with morphological and physiological properties typical of C. albicans (MacKenzie,

16

F. F. OGLETREE, A. T. ABDELAL AND D. G. AHEARN

1962; Joshi and Gavin, 1974; Evans, Odds and Holland, 1975; Dabrowa, Taxer and Howard, 1976). Recent studies reported the occurrence of atypical isolates of C. albicans which failed to produce germ tubes on initial isolation (Bowman and Ahearn, 1976; Crow, Bowman and Ahearn, 1977). The present study examines such isolates for their capacity to produce germ tubes.

MATERIALS AND METHODS Cultures. Yeast isolates and their sources are listed in Table 1. Culture G S U 30 demonstrated morphological and physiological properties typical of C. albicans and was employed as a control in all tests. All other G S U cultures failed to produce germ tubes and usually chlamydospores on initial screening with routine diagnostic procedures. Additional cultures selected on the basis of physiological similarities to C. albicans also were examined. Media. The following media were employed for germ-tube induction: glucose-beef-extract broth (GBE) containing 0 . 0 5 ~ glucose and 2.6Vobeef extract (Inolex); tissue culture medium 199 (TC 199, Difco); bovine serum (Gibco); bovine serum diluted 1:2.5 with sterile deionized water containing

Table 1. Cultures and source Isolate No.

Tentative identification

Source

GSU 1 1 GSU 9 GSU 21 GSU 22 GSU 23 GSU 24 GSU 25 GSU 26 GSU 27 GSU 28 GSU 29 GSU 30 GSU 33 CBS2 1912 CBS 1949 CBS 2312 CBS 6552 ATCC 3 28777

C. stellatoidea C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. langeronii C. claussenii C. tropicalis C. nouvelli C. viswanathii

urine feces blood vaginal secretions vaginal secretions 4 vaginal secretions 4 vaginal secretions vaginal secretions 4 vaginal secretions vaginal secretions vaginal secretions vaginal secretions 4 North Sea sputum unknown unknown animal origin, Paris Zoo cerebrospinal fluid

1 Georgia State University. 2 Centraalbureau voor Schimrnelcultures. 3 American Type Culture Collection. 4 Prolonged polyene therapy.

GERM-TUBE FORMATION BY ATYPICAL CANDIDA ALBICANS

17

20 pg/ml cysteine (Sigma Chemical) and 50 #g/ml sodium thioglycollate (Difco); amino acid medium (AAM) (Lee, Buckley and Campbell, 1975); and L-proline medium (Land et al., 1975a). The latter medium was also used with various modifications; adenosine, guanosine, cytosine and thymine (0.1 mM) were added or substituted for L-proline and increased concentrations of glucose (0.05 M-1.5 M) were used. Inoculum preparation. Yeasts were'grown on modified Sabouraud dextrose agar (MSD; 2.0 ~ glucose, 1.0 ~ neopeptone, and 2.0 ~ agar) for 24-48 h at 25 C. A 3-mm loopful of cells was used to inoculate flasks containing 100 ml Sabouraud dextrose broth (Difco) or yeast carbon base (Difco). These were incubated for 12-14 h on a gyratory shaker (200 rpm) at 25 C. The cells were washed three times and suspended in 10 ml of 8 mM phosphate-buffered saline, (PBS) pH 6.8 (Dulbecco and Vogt, 1954). Cell densities were adjusted to approximately 107 colony-forming units/ml from a standard curve made with GSU 30 relating optical density at 540 nm to colony-forming units/ml. Three tenths ml of the cell suspension was added to 2.7 ml of germ-tube-inducing medium (warmed to 37 C) to give a final cell concentration of approximately l06 cells/ml. All tests were transferred to an ice bath at the end of the incubation period prior to microscopic observation. If germ tubes were not observed at 3 h the test was repeated and incubation time extended to 7 h. Percent germ tubes was determined from the average of three separate enumerations of 200 cells. In temperature-shift experiments synchronous cultures of selected isolates (GSU 9, 21, 25, and 30) were obtained by the procedure of Land et al. (1975b). The cells were grown in MSD broth for 18-24 h, harvested, washed three times in PBS, resuspended in 50 ml of PBS and starved for 6 h on a gyratory shaker at 25 C. The cells were allowed to settle for 30 min and the supernatant was decanted and centrifuged at 100 • g for 5 rain in a 15~ mannitol solution. The supernatant was centrifuged at 1500 • g for 10 rain and the cell sediment was washed twice in PBS and resuspended to 107 cells/ml. Five ml of this cell suspension was added to flasks, warmed to 25 C and 39 C, each containing 45 ml of AAM. At 15 rain intervals up to 90 min, 5.0 ml was transferred from the flask at 39C to tubes in a 25C waterbath for 5 to 10 min (for rapid cooling) then placed on a roller drum at 25 C for the remainder of the 3 h incubation period. Effect of rifampin and amphotericin B on germ tubes. Rifampin (rifamycin SV, Sigma Chemical) and amphotericin B (Fungizone, Squibb) were added to the L-proline medium to determine their effects on germ-tube production by GSU 9 and GSU 30. Stock solutions of amphotericin B and rifampin were prepared by dissolving 10 mg in 1.0 ml of dimethyl sulfoxide (DMSO). These solutions were stored in the dark at 4C for no longer than one week prior to use. To determine minimal inhibitory concentrations (MIC), serial dilutions were made in Lproline medium to give concentrations of amphotericin B ranging from 0.05 to 100.0 pg/ml and of rifampin from 0.78 to 200 pg/ml. The tests were incubated in the dark on a roller drum at 25C for 48 h. Sublethal concentrations of

18

F. F. OGLETREE,A. T. ABDELALAND D. G. AnEARN

amphotericin B (0.003, 0.01, and 0.03/~g/ml) were used to increase cell permeability to rifampin. The effect of the two antimicrobial agents on germ-tube production and budding was determined with triplicate tests incubated in the dark on a roller drum at 25 C and 37 C. The yeasts incubated at 37 C were examined for germ tubes and cell viability at 3 and 20 h. Those at 25 C were shifted to 37C after 20 h and examined for germ tubes and viable cells 3 and 20 h later.

RESULTS In preliminary tests, C. albicans GSU 30, the typical isolate, produced consistently high numbers of germ tubes (greater than 75 ~) regardless of the medium used for growth of the inoculum. Inocula of 105 to l0 T cells gave approximately the same percentage of germ tubes in bovine serum but 106 cells/ml was critical for optimal germ tube formation in defined media. Incubation of cells for 10 to 14 h in yeast carbon base had little effect on subsequent germ-tube production by GSU 30, but similar treatment of atypical isolates reduced germtube production. Optimal germ-tube production for most isolates was obtained when cells were grown in SDB at 25 C for 18-24 h and when germ-tube induction media were warmed to 37C prior to inoculation. In initial screening of a few isolates no more than 40 ~ germ tubes were obtained but germ-tube production increased with maintenance in culture. No single germ-tube medium was optimal for all isolates, but most strains formed the highest percentage of germ tubes in bovine serum (Table 2). Frequently the germ tubes of atypical isolates were only a few/~m in length by 3 h (Fig. 1). The addition of cysteine and sodium thioglycollate to bovine serum enhanced germ-tube production by a few strains, particularly C. langeronii CBS 1912 and C. stellatoidea GSU 1. CBS 2312 produced predominately filamentous growth in SDB at 25 C with few blastospores (7-10 ~). At 37C in MBS approximately one-half of these blastospores formed germ tubes typical of C. albicans. Two isolates (C. viswanathii ATCC 28777, C. claussenii CBS 1949) did not

Fig. 1. Germ tubes ofGSU 9 after 3 h at 37C in modifiedbovine serum. Scaleequals 10/~m.

19

GERM-TUBE FORMATION BY ATYPICAL CANDIDA ALBICANS Table 2. Germ-tube production in various media ~ Isolate

Bovine serum

MBS

TC 199

L-proline

AAM

GBE broth

GSU 1 GSU 9 GSU 21 GSU 22 GSU 23 GSU 24 GSU 25 GSU 26 GSU 27 GSU 28 GSU 29 GSU 30 GSU 33 CBS 1912 CBS 1949 CBS 2312 CBS 6552 ATCC 28777

5 92 34 39 85 98 91 96 85 92 92 98 98 3 0 52 97 0

26 54 58 28 73 99 82 81 79 70 95 99 99 67 0 55 92 0

1 17 31 32 86 11 87 85 92 76 54 95 89 28 0 0 14 0

1 10 53 61 45 22 82 60 82 8 80 98 22 3 0 0 0 0

0 2 35 52 15 7 72 50 90 8 47 98 89 0 0 0 13 0

0 1 22 50 31 1 48 1 41 2 49 91 80 3 0 0 15 0

Percentage of cells bearing germ tubes, average of 3 counts of 200 cells. p r o d u c e germ tubes in a n y m e d i u m . All isolates except C. viswanathii A T C C 28777 were identified as C. albicans (Meyer, u n p u b l i s h e d D N A reassociation data). G l u c o s e c o n c e n t r a t i o n s above 2 ~ in L-proline m e d i u m reduced g e r m - t u b e p r o d u c t i o n by representative strains (Table 3). A d d i t i o n of p u r i n e a n d pyrimidine bases to the L-proline m e d i u m had n o a p p a r e n t effect on g e r m - t u b e prod u c t i o n by representative isolates. The time required for i n d u c t i o n o f germ tubes varied with the strain, t h e typical isolates s h o w i n g the shortest i n d u c t i o n period (Table 4). In an a t t e m p t to d e t e r m i n e whether the failure of G S U 9 to form germ tubes at 37 C reflects a different t e m p e r a t u r e - g r o w t h response, growth of G S U 30 and G S U 9 at Table 3. Effect of increasing concentrations of glucose on germ-tube production in L-proline medium Isolate No.

Glucose concentration (g/100 ml) 0 0.18 1 2

5

10

GSU 9 GSU 25 GSU 30

01 18 28

0 61 25

0 52 15

8 91 52

1Percent germ tubes after 3 h at 37C.

2 82 56

0 72 49

20

F , F.

OGLETREE,A. T. ABDELAL AND D. G . AHEARN

Table 4. Induction time for germ-tube production Isolate No.

Minutes at 39 C 15 30

45

60

90

150

GSU9 GSU 21 GSU 25 GSU30

01 1 1 86

0 60 32 86

0 85 40 89

0 81 72 88

0 84 75 87

0 8 15 85

Percent germ tubes per cell population determined after 150 min incubation at 25 C (following induction period) in amino acid medium.

different t e m p e r a t u r e s was e x a m i n e d in the g l u c o s e - b a s a l salt m e d i u m o f L a c r o u t e et al. (1965). T h e results s h o w the d o u b l i n g times to be essentially the s a m e in the two strains at 35 C a n d 25 C (Fig. 2). S i m i l a r results were also obt a i n e d at 3 0 C a n d 2 0 C . G S U 9 grew as a yeast at 4 0 C , b u t G S U 30 c o n v e r t e d to the h y p h a l stage at this t e m p e r a t u r e . .7

.6 9

,5 9 n

.4 " 0

.3 9

.2 9

.1 0

I

I

I

I

9

1

2

3

4

5

I

6

7

T I M E (HOURS)

Fig 2. Effect of temperature on growth of Candida albicans GSU 9 (25 C&, 35C/~ ) and GSU 30 (25C It 35C ~) in glucose-basal salts medium (Lacroute et al., 1965).

GERM-TUBE FORMATION BY ATYPICAL CANDIDA ALBICANS

21

Table 5. Effect of rifampin and amphotericin B on budding and germ-tube production by Candida albicans (GSU 30) after 3 hours in L-proline medium at 37 C. Rifampin/tg/ml

Amphotericin B (#g/ml) 0 0.003

0.01

0.03

0.8

0 1 5 10 20 30

8.931 +2 7.90 + 8.00 + 8.99 § 8.68 § 8.20 §

6.59 + 6.11 zk 6.30 • 5.85 • 6.49 • 6.41 •

7.15 + 5.93 • 5.91 5_ 5.66 • 5.15 • 5.67 i

4.41 - - - - - -

7.15 7.81 7.53 7.76 7.41 7.46

+ + § § § +

1 Log10 of colony-forming units/ml, average, triplicate tests, inocula l0 s cells. 2 (§ germ tubes well developed in > 8 0 ~ of cells, ( i ) germ-tube initials only in > 8 0 ~ of cells, ( - ) no germ tubes (cell lysis), ( - - ) not determined.

Concentrations of rifampin up to 250 /~g/ml did not appreciably inhibit budding (at 25 C) or germ-tube production (at 37 C) of G S U 30. The effect of amphotericin B up to 0.03 #g/ml was also negligible. The M I C of amphotericin B for G S U 30 in the L-proline medium was 0.4 #g/ml. Combinations of the two drugs, however, resulted in synergistic inhibition of budding and germ-tube development (Table 5). G e r m tubes produced by cells induced in the presence of rifampin and amphotericin B at concentrations of 0.01 and 0.03 pg/ml were only 3 4 / ~ m in length. Further development did not occur by 7 h. Cells exposed to both c o m p o u n d s for 20 h at 25 C did not initiate the formation o f new germ tubes when shifted to 37C. At 37C inhibition of budding was evident with combinations of rifampin and 0.003 #g/ml amphotericin B.

DISCUSSION Joshi et al. (1973b) and D a b r o w a et al. (1976), who studied isolates not specifically selected as atypical, indicated that the capacity to form germ tubes by C. albicans varied with the strain. The latter study included fifteen isolates, each of which produced m o r e than 1 6 ~ germ tubes in L-proline medium in comparison with media containing other single amino acids. F o r most strains TC 199 was more effective than the L-proline medium. Our atypical isolates always formed less than 6 0 ~ germ tubes and some gave fewer term tubes in T C 199 than in L-proline medium. Land et al. (1975b) who studied isolates which conformed to all biochemical and morphological criteria for C. albicans suggested that germ-tube formation correlated with a Crabtree-like effect, i.e., repression of mitochondrial activity. In our studies high concentrations of glucose in the L-proline medium (which would favor fermentation and repression of mitochondria) inhibited germ-tube production by both the typical

22

F. F. OGLETREE,A. T. ABDELALAND D. G. AHEARN

and the atypical strains. Glucose-beef-extract broth which functioned satisfactorily (>95~o accuracy) in routine screening of clinical isolates for C. albicans (Bowman and Ahearn, 1975) was unsatisfactory for certain atypical isolates. A few atypical strains were induced to form germ tubes in bovine serum by the addition of either thioglycollate and cysteine or both, but these reducing agents gave no appreciable effect in defined media (L-proline, AAM or TC 199). Contradictory results on the effects of cysteine on germ-tube production have been reported (Wain, Price and Cawson, 1975; Dabrowa et al., 1976). In agreement with Evans et al. (1975), temperature appeared to be a major factor in the dimorphism of C. albicans, and as exemplified by GSU 9, the importance of medium composition varied with the strain. Medoff et al. (1972) showed that amphotericin B potentiated the antifungal effect of rifampin in yeasts. Apparently, sublethal concentrations of amphotericin B increased cell-membrane permeability allowing increased uptake of rifampin by the cells. Beggs, Sarosi and Walker, (1976), demonstrated synergistic inhibition of Candida sp. with combinations of rifampin and amphotericin B as low as 25 and 0.1 #g/ml respectively. Consistent with these results we found that concentrations of rifampin as low as 1 #g/ml in combination with 0.01 to 0.03 #g/ml amphotericin B inhibited budding and germ-tube maturation of unstarved cells of GSU 30, but not germ-tube initiation. Germ tubes developing in the presence of rifampin and the subinhibitory levels of amphotericin B were no longer than 4/~m. Rifampin (rifamycin SV) has been shown to bind specifically to mitochondrial RNA polymerase (Scragg, 1976) but not to inhibit nuclear RNA polymerase in Saccharomyces cerevisiae (Adman, Schultz and Hall, 1972). This suggests that mitochondrial RNA polymerase function is more necessary for budding and germ-tube maturation than germ-tube initiation. Ahearn et al. (1966) suggested that the percent of germ tubes formed by a given strain was primarily an inherent capacity of the strain rather than a dependency upon specific metabolites in the induction medium. The variability in germ-tube production between different strains may be a reflection of the size of their pool of mitochondrial RNA polymerase. Dabrowa et al. (1976) have reviewed the discrepancies reported by various authors on factors affecting morphogenesis of C. albicans. Our atypical strains showed an even greater divergence in their potential for germ-tube formation. In our experience atypical isolates are occurring more frequently in the clinical laboratory, often from patients who have received extensive chemotherapy for candidiasis. In other studies (unpublished data) representative atypical isolates have been found to be as virulent or more virulent for mice than typical isolates. The need to detect these isolates in the clinical laboratory is increasingly important. In general, bovine serum appears to be the most suitable medium for diagnosis of variant strains of C. albicans. Received 18 October 1977

GERM-TUBE FORMATION BY ATYPICAL CANDIDA ALBICANS

23

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F. F. OGLETREE, A. T. ABDELAL AND D. G. AHEARN

SCRAGG,A. H. 1976. The isolation and properties of a DNA-directed RNA polymerase from yeast mitochondria. - - Biochim. Biophys. Acta 442:331-342. STENDERUP,A. and TnOMSEN, J. B. 1964. Identification of C a n d i d a albicans. - - Acta Pathol. Microbiol. Scand. 62: 303- 304. TASCHDJIAN,C. L., BURCHALL,J. and KOZINN, P. J. 1960. Rapid identification of C a n d i d a albicans by filamentation on serum and serum substitutes. - - Am. J. Dis. Child. 99: 212215. WAIN, W. H., PRICE, M. F. and CAWSON,R. A. 1975. A re-evaluation of the effect of cysteine on C a n d i d a albicans. - - Sabouraudia 12: 74-82.