Rate of Arabinitol Production by Pathogenic Yeast Species

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JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1981, p. 189-194 0095-1137/81/080189-06$02.00/0 Vol. 14, No. 2 Rate of Arabinitol Production by Pathogenic Yeast Species EDWARD M. BERNARD,* KERYN J.

1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1981, p /81/ $02.00/0 Vol. 14, No. 2 Rate of Arabinitol Production by Pathogenic Yeast Species EDWARD M. BERNARD,* KERYN J. CHRISTIANSEN,t SO-FAI TSANG, TIMOTHY E. KIEHN, AND DONALD ARMSTRONG Diagnostic Microbiology Laboratory, Memorial Hospital and Infectious Disease Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York Received 30 December 1980/Accepted 2 April 1981 D-Arabinitol is a five-carbon polyol that is produced by many fungi. Detection of the metabolite has been reported in serum from patients with invasive candidiasis. We studied the production and assimilation of arabinitol by 46 clinical isolates of yeast species. Cultures of isolates of Candida albicans (9 strains), Candida tropicalis (12 strains), Candida parapsilosis (13 strains), Candida krusei (4 strains), Candida pseudotropicalis (3 strains), Torulopsis glabrata (3 strains), and Cryptococcus neoformans (2 strains) were assayed by gas-liquid chromatography. Yeast cells were cultured at 340C in yeast nitrogen base with 3.0 g of glucose per liter. At 1.5- to 3-h intervals, cells were counted and glucose and arabinitol were measured in media filtrates. The levels of arabinitol in cultures with 7.5 x 106 yeast cells per ml were compared. The mean concentrations of the metabolite in C. albicans, C. tropicalis, C. parapsilosis, and C. pseudotropicalis cultures were 14.1, 1.6, 8.4, and 5.5 fig/ml, respectively. No arabinitol was detected in cultures of C. krusei, T. glabrata, or C. neoformans. D-Arabinitol is a five-carbon sugar alcohol or polyol; it is produced by pathogenic yeast species, and elevated levels of the metabolite are present in sera from patients with invasive candidiasis (5). The purpose of this study was to determine how much arabinitol was formed in cultures of clinical isolates of yeasts and what conditions favored its formation. Polyols are major constituents of many fungi (7, 11). Glycerol, a three-carbon polyol, was first described as a product of yeast metabolism by Pasteur. Osmophilic yeast species recovered from honey, brood comb pollen, and flower parts have been found to produce glycerol and erythritol (12). The major polyol detected in cultures of these isolates, however, was arabinitol: as much as 60% of the sugar utilized by these strains was converted to arabinitol. Arabinitol had earlier been detected in the fermentation residue of bakers' yeast and blackstrap cane molasses (1). Candida tropicalis and Candida lipolytica have been reported to produce arabinitol when grown on sugars or n-alkane mixtures (4). One strain of C. tropicalis afforded a yield of 50% based on the weight of dissimilated alkane. The remarkable efficiency with which yeasts convert diverse carbon sources to D-arabinitol has been the subject of several patents. t Present address: Royal Perth Hospital, Perth, Australia. 189 Our interest in polyols developed in the course of evaluating the use of gas-liquid chromatography in the diagnosis of disseminated candidiasis. It was reported that patients with fungemia or candidiasis had high serum levels of substances containing mannose (8). We were unable to confirm this, but found that a major component of yeast cultures was present in sera from some patients with invasive infection due to Candida species (5). We identified the yeast metabolite as D-arabinitol by mass spectrometry, mixed melting point determination, and by cochromatography studies. D-Arabinitol is not known to be a normal human metabolite (14); it has been detected in low levels in sera from uremic patients and in urine from healthy individuals (9). It is not known if arabinitol detected in urine derives from diet constituents, is a product of host metabolism, or is produced by yeast resident in the bowel. The route of elimination and the clearance rate have also not been described. The present study was undertaken to determine which yeast species produce arabinitol. We also tested the ability of different strains to assimilate D- or L-arabinitol and examined the relationship between arabinitol production and osmophilicity. (This work was presented in part at the 80th Annual Meeting of the American Society for Microbiology, Miami, Fla., May, 1980.)

190 BERNARD ET AL. MATERIALS AND METHODS Yeast isolates. Forty-three yeast strains were tested. The species and number of isolates per species are listed in Table 1.

2 190 BERNARD ET AL. MATERIALS AND METHODS Yeast isolates. Forty-three yeast strains were tested. The species and number of isolates per species are listed in Table 1. Isolates were recovered from clinical specimens submitted to the microbiology laboratory of the Memorial Sloan-Kettering Cancer Center. The prevalence of yeasts and methods of identification used at Memorial Hospital were recently reported (6). Isolates were maintained on Sabouraud dextrose agar slants. Candida albicans strains AP/ F23 and AP/H09 were recovered from autopsy cultures. Culture conditions. The medium employed in studies of the rate of arabinitol formation was Yeast Nitrogen Base broth (YNB) with glucose as the sole carbon source at 3.0 g/liter (YNB plus glucose). The media was dispensed in 15-ml plastic centrifuge tubes. Cultures were placed at 34 or 37 C in a shaking incubator (New Brunswick Scientific Co., New Brunswick, N.J.) set at 150 rpm. Inocula were prepared by culturing the isolate at 34 C in YNB plus glucose for 36 h; the cell number per ml was determined as described below, and the culture was diluted in saline to give 2 x 105 cells per ml. A portion of this, 0.40 ml, was added to 7.6 ml of YNB plus glucose to yield an initial concentration of 104 yeast cells per ml. Solid media for assimilation studies were prepared by adding filter-sterilized solutions of D-glucose, D- arabinitol, or L-arabinitol in water to YNB and 3% Noble agar. The solutions of the pentitol enantiomers and the sugar were added to the sterile, melted agar to give 3.0 g/liter. The agar (25 ml) was poured into square petri dishes (100 by 15 mm), and inocula, prepared as described above, were applied by using a Steers replicator. Cell counts. The number of yeast cells per ml of culture was determined by using a hemacytometer. At least 100 cells (all cells, including well-formed buds) were counted. The number of colony-forming units was determined by using a pour-plate technique. Serial 10-fold dilutions of cultures were made in sterile saline, and 1.0 ml was mixed with 10 ml of sterile Trypticase soy agar and poured into plastic petri dishes (100 by 15 mm). The number of colony-forming units per ml was calculated after 18 to 36 h of incubation at 34 C, by counting plates with 35 to 350 colonies and multiplying the value obtained by the reciprocal of the TABLE 1. Arabinitol production and rate ofgrowth ofyeast isolates Mean No. of genera- Mean arabinitol Organism strains tion cnn(gm) tested time concn (pg/ml)' (h) C. albicans ( )h C. tropicalis ( ) C. parapsilosis ( ) C. pseudotropicalis ( ) C. krusei C. neoformans T. glabrata acalculated for 7.5 x 106 cells per ml. brange given within parentheses. J. CLIN. MICROBIOL. dilution factor. Sampling. After incubation for 4 to 8 h, cells were counted directly at 1- to 4-h intervals. Cultures were blended in a Vortex mixer, and 1.0 ml was removed with a sterile 5-ml syringe. The hemacytometer was loaded by touching a drop to the edge of the cover slip; the remainder was used to determine the number of colony-forming units or, by using a 0.45-gm membrane filter (Millipore Corp.) after filtration, the concentrations of glucose or arabinitol. Samples for carbohydrate analysis were stored at -20'C. Analytical. The trimethylsilylethers of carbohydrates were measured by using gas-liquid chromatography as previously described (5). The derivation procedure and analysis are based on the method of Sweeley et al. (13). The internal standard for arabinitol determinations, a-methylmannoside in acetone (5.0 jig/ml, 0.20 ml), was added to 0.10 ml of culture filtrate in a glass centrifuge tube. This was blended in a Vortex mixer and evaporated under a nitrogen stream. A ml portion of the derivatizing agent (pyridine-hexamethyldisilazane-trimethylsilyl chloride, 6:2:1 [vol/ vol]) was added to the residue, and the tube was sealed with a Teflon-lined cap, placed in a 45 C water bath, and agitated for 15 min. After a brief centrifugation, 5 pl of the supernatant was analyzed by gas-liquid chromatography with a flame-ionization detector and an oven temperature program (140 to 220 C, 4 per min). The Hewlett-Packard 5730A gas chromatograph was equipped with a 3380A integrator. The instrument was fitted with dual 1.8-m, coiled glass columns (62.5 mm in diameter) packed with 3% SE-30 on 80/100 mesh Gas-Chrom Q (Hewlett-Packard, Avondale, Pa.). The nitrogen carrier flow was set at a rate of 40 ml/min. The detector response factor of arabinitol relative to a-methylmannoside was 1.33 as determined by analysis of mixtures of known amounts of the polyol and the standard in water. When glucose and arabinitol were measured simultaneously, mannitol at 50 yg/ml was incorporated in the internal standard solution. The glucose level was derived by dividing the product of the known mannitol concentration and the sum of peak areas of the alpha and beta anomers of glucose by the peak area of mannitol. Media, chemicals, and solvents. YNB, glucose, and Noble agar were obtained from Difco Laboratories, Detroit, Mich. Trypticase soy agar was from BBL Microbiology Systems, Cockeysville, Md. Hexamethyldisilazane, trimethylsilyl chloride, a-methylmannoside, and pyridine were purchased from Aldrich Chemical Co., Milwaukee, Wis. D-Arabinitol, L-arabinitol, and mannitol were from Applied Science Laboratories, State College, Pa. Reagent grade acetone from Fisher Scientific Co., Pittsburgh, Pa. was used. RESULTS Figure 1 shows a chromatogram of a culture filtrate of C. albicans strain AP/F23. The peak with a retention time of 8.62 min was not present in chromatograms of uninoculated media. The peaks at 9.51, 12.11, and min are, respectively, from the internal standard and the alpha and beta anomers of glucose. The substance

VOL. 14, 1981 w cz R g-. 0, 4n cq 4 O 5 10 15 RETENTION TIME (MIN) FIG. 1. Gas-liquid chromatography of the trimethylsilyl ether derivative of C. albicans strain AP/F23 filtrate.

3 VOL. 14, 1981 w cz R g-. 0, 4n cq 4 O RETENTION TIME (MIN) FIG. 1. Gas-liquid chromatography of the trimethylsilyl ether derivative of C. albicans strain AP/F23 filtrate. D-Arabinitol and the internal standard have retention times of8.62 and 9.51 min, respectively. The major glucose anomers are at and min. eluting at 8.62 min was collected and submitted for mass spectrometry (model 3300, Finnigan Corp., Sunnyvale, Califl. Chemical ionization mass spectrometry (methane) of this material yielded a pseudomolecular ion at a mass-tocharge ratio of 513 (Fig. 2). Similar analysis of an underivatized sample and the major ion fragments produced by electron beam ionization identified the compound as a pentitol. Cochromatography studies and mixed melting point determinations of the peracetate derivative demonstrated that the yeast metabolite was D- arabinitol. In early experiments to determine which yeast species produced this metabolite and at what rate they produced it, isolates were cultured in YNB plus glucose and the arabinitol level in filtrates was measured after 24 and 48 h of incubation. Figure 3 shows the number of yeast cells per ml and the glucose and arabinitol levels obtained by sampling a culture at more frequent intervals. ARABINITOL PRODUCTION BY YEASTS 191 The C. albicans strain AP/F23 used was recovered from tissues obtained at autopsy from a patient with disseminated infection; the yeast was grown in 2.5 mg of glucose per ml in YNB at 37 C. These early studies showed that by sampling cultures which had been in stationary phase for indeterminate periods, the peak of the arabinitol curve might be missed. The decrease of the metabolite in filtrates as the culture reached stationary phase was examined by determining the relationship between intracellular and extracellular arabinitol levels. With C. albicans strain AP/H09 and the same conditions as above, we sampled the cultures at frequent intervals: a portion was ultrasonically disrupted, an undisturbed portion was counted, and arabinitol was measured in the sonicate and culture filtrate. Samples were disrupted in a Branson sonifier for 15 min at 100 W (Heat Systems, Plainview, N.Y.). Figure 4 illustrates the results of this experiment. The difference between arabinitol concentrations of the cellfree media and the sonic extract of the culture represents the amount released as the yeast cell wall was disrupted. Figure 4 shows that after 40 h of incubation the arabinitol concentration in the culture filtrate, i.e., the extracellular concentration, was 149,ug/ml. Sonication of a portion of the culture released 61,ug/ml of arabinitol. As the culture remains in stationary phase, there is a decrease in both intracellular and extracellular levels of the metabolite. The intracellular concentration of arabinitol can be derived from the amount released by ultrasonic disruption of a yeast suspension. In Fig. 4, at 40 h, the cell number was 107 per ml. The mean diameter of the cells, measured with a microscope with a calibrated eyepiece, was 3 p,m. This gave a total intracellular volume of about 1/500 of the culture volume. Multiplying the reciprocal of this factor by the amount of arabinitol released, 61 ug/ml, gave an intracellular concentration of about 30 mg/ml. A basis for comparing arabinitol production rates of various strains was established by defining a single point during the exponential growth phase and determining the arabinitol level in cultures at that reference point. A cell concentration of 7.5 x 106 yeast cells per ml was chosen as the reference because preliminary studies indicated that cultures with this cell number were increasing exponentially and arabinitol levels were sufficient to allow accurate quantitation. Isolates of yeast species were grown in YNB plus glucose at 340C with agitation. Freshly prepared inocula were used to give an initial concentration of 100 yeast cells per ml. After 8 to 12 h of incubation, cultures were sampled at 1.5- to

4 192 BERNARD ET AL. 100 J. CLIN. MICROBIOL. - c- co al) CZ ct Ca) CL) L) 4..I. I. Oa.I, II ~~513 IL ' m/e 5O0 S m/e FIG. 2. Chemical ionization (methane) mass spectrum of the trimethylsilyl derivative of arabinitol. The pseudomolecular ion is present at 513 m/e. The compound was isolated from a culture filtrate of C. albicans strain AP/F < 2000L T :3 "I 6 (n z O 1500 D 5 z 1000 FIG. 3. The kinetics ofgrowth, glucose utilization, and arabinitol production of C. albicans strain API F23 are shown. The media was YNB plus glucose as the sole carbon source. 3-h intervals. Levels of arabinitol in the filtrates of cultures with between 2.5 x 106 and 1.2 x 107 yeast cells per ml were used to derive, by linear extrapolation, the arabinitol concentration at the reference point. For example, a C. albicans culture after 18 h of incubation was found to O 4 0 Z U, O0 0 2 L TIME (HOURS) FIG. 4. Comparison of arabinitol levels in filtrate and sonic extract of C. albicnas strain AP/H09. The decrease of arabinitol levels in the culture filtrate over time indicates metabolism by stationary phase cells. The differences between arabinitol levels in the sonic extract and filtrate are a measure of intracellular concentrations. contain 5.62 x 106 yeast cells per ml, and 2 h later, the cell numbers had increased to 1.10 x 107; the corresponding arabinitol levels were 15.0 ug/ml and 28.2 jg/ml. On a graph of cell number I

VOL. 14, 1981 versus arabinitol concentration, the line defined by these values was plotted, and the arabinitol concentration at 7.5 x 106 yeast cells per ml was determined to be 18.0,ug/ml.

5 VOL. 14, 1981 versus arabinitol concentration, the line defined by these values was plotted, and the arabinitol concentration at 7.5 x 106 yeast cells per ml was determined to be 18.0,ug/ml. Generation times were also calculated at this interval from the relationship: g = 0.69t/(ln X - X0), where t is time between samplings and X0 and X are the cell numbers at initial and subsequent samplings respectively (3). A total of 46 yeast strains were tested. Table 1 presents the number of strains of each species, the mean generation times and the mean and range of arabinitol concentrations. All of the strains of C. albicans, C. tropicalis, Candida parapsilosis, and Candida pseudotropicalis tested produced D-arabinitol. Production rates, as measured by the concentration of the metabolite at the reference point, varied over a wide range. Among species which produced arabinitol, mean concentrations are of similar magnitude. None of the strains of Candida krusei, C. neoformans, and Torulopsis glabrata produced arabinitol. The ability of tested strains to assimilate D- arabinitol and to grow in the presence of high sugar concentrations is shown in Table 2. None of the 46 isolates assimilated L-arabinitol. Only producer strains assimilated D-arabinitol. Every C. tropicalis strain tested grew on a media incorporating D-arabinitol as the sole carbon source; growth was apparent in most cases after incubation at 340C for 24 h. Only 3 of the 13 strains of C. parapsilosis assimilated the pentitol; growth was slow and sparse. Sugar tolerance was a feature of the majority of isolates; producer and nonproducer strains grew in the presence of 60% glucose. DISCUSSION D-Arabinitol is a major product formed in cultures of some yeast species. Arabinitol is me- TABLE 2. Growth of isolates on D-arabinitol and in the presence of a high concentration ofglucose No. which grew on agar with: No. of Organism strains 1% D-ar- 60% glutested abinit.ol, cose a YNB yeast extractb C. albicans C. tropicalis C. parapsilosis C. pseudotropicalis C. krusei C. neoformans T. glabrata a Incubated at 340 for 48 h. bread after 7 days at 34 C. ARABINITOL PRODUCTION BY YEASTS 193 tabolized by some strains; as glucose is exhausted, the metabolite is taken up by cells and utilized. The high intracellular concentrations and the efficiency of arabinitol production and utilization suggest that the compound may serve an important physiological function and may define in significant ways the intracellular millieu of the yeast. Investigations of the functional significance of fungal polyols were reported in studies with Saccharomyces cerevisiae and Saccharomyces rouxii (2). The former organism is nonosmophilic and the latter thrives in media with high solute concentrations. Osmophilic strains differed from sugar-intolerant strains in their intracellular composition: osmophilic strains produced large amounts of arabinitol and other polyols. The activity of many enzymes diminishes with increasing levels of certain solutes. The halophilic bacteria maintain functional integrity at high osmotic pressure by concentrating K+ at the expense of Na+. The yeasts which tolerate concentrated sugar solutions may, in an analogous manner, modulate the flux of solutes across the cell membrane by converting glucose to arabinitol and concentrating the polyol inside the cell. Arabinitol and other polyols are, like potassium, compatible solutes. They may be present at high concentrations without inhibiting enzyme activity. Among the yeast species tested, no clear relationship exists between the ability to produce arabinitol and sugar tolerance. Every strain of T. glabrata, for example, grew on a media with 60% glucose; none of these isolates produced arabinitol. Further study is needed to determine if nonproducer strains that tolerate high solute levels elaborate other polyols. Only strains that produced arabinitol were able to grow in culture when D-arabinitol was the sole carbon source. Ability to assimilate this isomer was not present in all producing strains, and, as a consequence, the property is not useful in predicting significant production by an isolate. The observation that L-arabinitol is not utilized by any strain supports the assignment of the configuration given to the pentitol detected in cultures. The relevance of these studies to the use of serum or urine arabinitol levels in the diagnosis of invasive candidiasis is twofold. First, arabinitol was produced by every strain of the Candida species most frequently isolated from blood or tissues at this hospital: C. albicans, C. tropicalis, and C. parapsilosis. A second point which these studies serve to emphasize is that arabinitol is a metabolite. In culture the amount of arabinitol formed and the rate of its formation depend on

194 BERNARD ET AL. cell number, the efficiency of the strain, the rate of growth, and the availability of substrate. These factors are also likely to influence arabinitol production in vivo.

6 194 BERNARD ET AL. cell number, the efficiency of the strain, the rate of growth, and the availability of substrate. These factors are also likely to influence arabinitol production in vivo. Detection of yeast metabolites is one of many efforts to improve the diagnosis of life-threatening fungal infections. Elevated levels of arabinitol, a major product of some Candida species, have been detected in serum from patients with proven infection (5, 10). We undertook this study, in part, to see if yeasts elaborate arabinitol in sufficient quantity to account for its accumulation in vivo. The levels in serum or urine that might be found as a result of an infection depend on the efficiency of arabinitol production in vivo and on the mode and rate of elimination of the metabolite by the host. We are currently investigating the metabolism and excretion of D-arabinitol by experimental animals and man. ACKNOWLEDGMENTS This work was supported in part by a Memorial Hospital grant from the Special Projects Committee of the Memorial Society of Memorial Hospital. Mass spectrometry was performed by V. Parmakovitch, Chemistry Department, Columbia University, New York. Thanks are due Fitzroy Edwards of the Microbiology Laboratory and Brian Wong and Jonathan Gold of the Infectious Disease Service for advice, assistance, and criticism. LITERATURE CITED 1. Binkley, W. W., and M. L. Wolfrom Chromatographic fractionation of cane blackstrap molasses and its fermentation residue. J. Am. Chem. Soc. 72:4778- J. CLIN. MICROBIOL Brown, A. D Microbial water relations: features of the intracellular composition of sugar-tolerant yeasts. J. Bacteriol. 118: Doelle, H. W Bacterial metabolism, 2nd ed. Academic Press, Inc., New York. 4. Hattori, L., and T. Suzuki Microbial production of D-arabinitol by n-alkane-grown Candida tropicalis. Agric. Biol. Chem. 38: Kiehn, T. E., E. M. Bernard, J. W. M. Gold, and D. Armstrong Candidiasis: detection by gas-liquid chromatography of D-arabinitol, a fungal metabolite, in human serum. Science. 206: Kiehn, T. E., F. F. Edwards, and D. Armstrong The prevalence of yeasts in clinical specimens from cancer patients. Am. J. Clin. Pathol. 73: Lewis, D. H., and D. C. Smith Sugar alcohols (polyols) in fungi and green plants. New Phytol. 66: Miller, G. G., M. W. Witwer, A. I. Braude, and C. E. Davis Rapid identification of Candida albicans septicemia in man by gas-liquid chromatography. J. Clin. Invest. 58: Pitkanen, E The serum polyol pattern and the urinary polyol excretion in diabetic and in uremic patients. Clin. Chim. Acta. 38: Roboz, J., Suzuki, R., and J. F. Holland Quantification of arabinitol in serum by selected ion monitoring as a diagnostic technique in invasive candidiasis. J. Clin. Microbiol. 12: Spencer, J. F. T Production of polyhydric alcohols by yeasts. Progr. Ind. Microbiol. 7: Spencer, J. F. T., and H. R. Sallans Production of polyhydric alcohols by osmophilic yeasts. Can. J. Microbiol. 2: Sweeley, C. C., R. Bentley, M. Makita, and W. W. Wells Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. J. Am. Chem. Soc. 85: Touster, O., and D. R. D. Shaw Biochemistry of acyclic polyols. Physiol. Rev. 42:

Erythritol Production by a Yeastlike Fungus

APPLIED MICROBIOLOGY Vol. 12 No. 3 p. May, 1964 Copyright 1964 American Society for Microbiology Printed in U.S.A. Erythritol Production by a Yeastlike Fungus G. J. HAJNY, J. H. SMITH 1, AND J. C. GARVER