Sterol content and polyene antibiotic resistance in isolates of Candida krusei, Candida parakrusei, and Candida tropicalis L. M . SAFEA N D S. H . SAFE Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
D L J P ( I ~ / I ?(?f I(C , I /I I/ L ~ I ? I ~U~li~.ersi/?. J/I?, o f G ~ ~ ~ , lGrrelpll, ph, 011/., C O I I ( I ~ ~ I AND
R. E . SUBDEN A N D D. C. MORRIS Depot~tt?~etlt of G e r ~ e t i cU/li~.ersit.v ~, of G~relph,Grtelpl~,Orlt., Cnrlnrlo Accepted December 20, 1976
SAFE.L. M., S. H. SAFE,R. E. SUBDEN, and D. C. MORRIS.1977. Sterol content and polyene Cotldidrl pcrroLrlrsri, and Ctrtlditlrr antibiotic resistance in isolates of Co~ididaXr~~sei. /ropic~olis.Can. J . Microbiol. 23: 39W01. Three isolates, one from each species of Corididl~L-r~rsei,C . pnrokrirsei, and C . tropicolis, obtained from infected patients, were more tolerant of significantly higher concentrations of polyene antibiotics than the corresponding reference wild types. The resistant strains isolated had the same sterols as their wild-type counterparts but in lowerconcentrations. SAFE,L. M., S. H. SAFE,R. E. SUBDEN et D. C. MORRIS.1977. Sterol content and polyene antibiotic resistance in isolates of Candida knrsei, Candida parakrrrsei, and Candidn rropicalis. Can. J . Microbiol. 23: 398-401. Trois isolats, un de chaque espece de Candida krrrsei, C. parakrirsei et C. tropicalis, isolCs de patients infect&, tolerent de f a ~ o nsignificative des concentrations plus elevkes d'antibiotiques polyenes que les souches sauvages correspondantes utilisees comme timoins. Les .ouches resistantes isolees ont les memes stCrols que les types sauvages, mais en concentrations plus faibles. [Traduit par le journal]
Introduction I t has been previously reported that Candida can he routinely mutated to polyene antibiotic resistance under laboratory conditions (1, 6). These mutant strains may be either single gene mutations (8, 21) involving discrete lesions in the sterol biosynthetic pathway (2, 18) or multistep variants from training experiments using serial transfers through solid media containing increasing concentrations of the antibiotic (10, 11, 15, 19). Although polyene antibiotics have been used therapeutically for 15 years, reports of clinically isolated polyene-resistant strains of Candida are surprisingly few (9, 22). For some reason there has been n o selection for antibiotic-resistant Candida under present clinical procedures. In this study, 864 isolates of Candida, obtained from patients at the Toronto Children's Hospital, were given a preliminary screening for polyene sensitivity on Szybalski (3) plates. Three polyeneresistant strains were isolated. Taxonomic studies indicated that the resistant strains were C. krusei, C. parakrusei, and C. tropicalis. This
paper reports on the sterol content of the resistant strains, reference wild-type or sensitive strains, and then suggests a possible correlation between the sterol content and polyene resistance.
Materials and Methods Strains and Crrlture Cot~rlitiot~s The wild-type C. olbicans ATCC 24433 was obtained from the American Type Culture Collection. Reference wild-type cultures of C. krrrsei, C . porakrrwei, and C. tropicolis were obtained from D r . P. Fleming, Department of Bacteriology, Toronto Children's Hospital (TCH). Resistant strain C. krrrsei TCH-T2672 was isolated from the urine of a 17-month-old male admitted for correction of a bladder extrophy. The patient had received nystatin treatment G times before the isolation of this strain. Cntldido parokrrrsei TCH-T1 1 21 was isolated from the urine of a 9-month-old female admitted for gastroenteritis and otitis media. She had received ampicillin and trimethoprim-sulphamethaoxzole but no polyene antibiotic. Cottdirla tropicalis TCH-S1163 was isolated from the stool of a 10-year-old male admitted for idiopathic acquired pancytopenia. He had received nystatin treatment for 4 d before isolation of this strain. Unless otherwise specified, wild-type cultures were kept on yeast propagation medium (YPM) consisting of 0.3% malt extract, 0.3% yeast extract, 0.5% peptone,
SAFE E T AL.
TABLE1. Minimum inhibitory concentrations of amphotericin B, candicidin, nystatin, and pimaricin for wild-type and resistant strains of C. krusei, C. parakrrrsei, C. fropicalis, and C. albicarls -
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
Strain
Candida krlcsei (wild type) C. krusei-R (isolate TCH-T2672) C. parakrusei (wild type) C. parakr~rsei-R (isolate TCH-TI 121) C. tropicalis (wild type) C. tropicalis-R (isolate TCH-S1163) C , albicans (wild type ATCC 24433)
Amphotericin B, ~g/ml
-
Candicidin, Nystatin, ~g/ml dm1
-
Pimaricin, ~g/ml
0.6
0.4
4.0
3.5
70.0
3.0
15.0
5.0
1.5
0.5
4.0
3.0
20.0
2.0
5.0
5.0
2.0
0.8
8 .O
5.0
25.0
2.0
10.0
5.0
0.5
0.3
3.0
3.5
1.0% glucose, and where necessary 1.5% agar. Resistant strains were kept on YPM plus 10 ug/ml nystatin.
Polyetle Antibiotics Antibiotics were stored at - 17°C till immediately before use. Stock solutions of amphotericin B (1 mg/ml), candicidin (1 mg/ml), and nystatin (10 mg/ml) were prepared in dimethylsulphoxide (DMSO). Piniaricin (I mg/ml) was dissolved in propylene glycol. Freshly prepared solutions of antibiotics were added to the medium after it had cooled to 60°C after autoclaving. Sensitivity Tests As a preliminary screening procedure, 4 of the 864 strains isolated plus a wild-type control (C. nlbicnrls ATTC 24433) were streaked on each plate. A suspension of each strain was made in 0.14 M saline One loopful of each suspension was streaked on a Szybalski (3) gradient plate at 200 ~ g / m nystatin. l Square (100 x 15 mni) petri plates were used for all tests. Cultures were incubated at 37'C and growth scored after 48 h. The minimum inhibitory concentrations (MIC) were determined by plate-dilution tests. At first the antibiotics l were incorporated Into the medium at 0.5 ~ g / mincrements from 0.0 to 100.0 g/ml. Succeeding experiments used 0.02 ~ g / m lincrements over a narrower range. Aliquots containing 0.1 ml of 2 x l o 5 cells/ml in 0.14 M sallne were spread uniformly on plates containing the antibiotics. Sepnrafion ntld Idetltificatiot~of Stet 01s Forty-eight hour, air-agitated (bubblers), 3-! cultures of Cnndida were harvested by centrifugation, washed twice with 0.14 M saline, and lyophilized. The cells were then macerated and refluxed in 300 ml of 10% K O H in methanol for 2 h. Cell wall fragments and other debris were removed by filtrat~onthen washed with ether. The filtrate was diluted with 2 volumes of water and extracted with 2 volunies of diethyl ether. The ether phase was washed wlth water, dried over Na,SO,, concentrated to dryness in a flash evaporator, resuspended in a small volume of benzene, and applied to a preparative thinlayer plate (silica gel H F 254 + 366) then developed with
hexane:ether:ethanol (80:20:2). Sterols detected by UV quenching were removed from the plate, eluted from the silica gel, and converted into their corresponding acetate derivatives with acetic anhydride pyridine (2: 1). Steryl acetates were purified by preparative thin-layer chromatography as described above and analyzed on HewlettPackard 700 and Beckman GC72 gas chronlatographs using 8 ft x & in. glass columns packed with 1.5% OV17 on Gas Chrom. Q at 2605C. The carrier gas was helium at 60 ml/min and the detector and inlet temperatures were 290°C. Internal standards for sterol identification were obtained from a previous study (18).
Results and Discussion Of 864 isolates tentatively identified as Ccrtidiu'u and screened for polyene sensitivity, three demonstrated unusual resistauce to polyene antibiotics on Szybalski plate gradients. Further taxonomic tests including germ-tube formation, fermentation reactions, and growth on corn meal Tween 80 media according to Matheson (17) indicated that strain TCH-T2672 was C. tropicalis, TCH-TI 12 1 was C. paraknrsei, and TCHS1163 was C. knrsei. Polyene MIC for each of these isolated strains were compared to their wild types as shown in Table 1. The data indicate that the wild-type strains were more sensitive to all of the antibiotics used and that candicidin was more potent than pimaricin. It should also be noted that the difference in MIC for all resistant strains was greatest for amphotericin, and least for pirnaricin. In fact, C. tropicnlis, TCH-S 1 163, and wild-type C. tropicalis had identical MIC's for pirnaricin. In a previous report, Hamilton-Miller (7) identified two strains
CAN. J.
MICROBIOL. VOL.
23, 1977
TABLE2. Sterol content of various Candida strains expressed as pg sterol/mg dry weight
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
Candida krusei
Candida parakrusei
Candida fropicalis
Sterol*
Rct
Wild type
Resistant (TCH-T2672)
Wild type
Resistant (TCH-TI 121)
Wild type
Lichesterol Ergosterol Fecosterol Unknown sterols
77/20 82/20 91/20 99/20 91/20 l05/20
53 .O 210.0 320.0 -
6.0 4.0 2.0 9.0
62.0 920.0 20.0
89.0 360.0 14.0 36.0 -
11 . O 50.0 1.1 -
-
1 .O 28.0 1 .O 5.6 0.6 2.0
583.0
21 .O
1002.0
38.2
499.0
62.1
-
Resistant (TCH-S1163)
'Identified by comparison with authentic standards (16). tRetention time on gas chromatographs 1.5% OV-17, 260°C, relative to internal standard cholestane.
of C. krusei out of 413 clinically isolated yeasts. One had an MIC of 2 pg/ml and the other 16 pg/ml. Further work with other strains showed that C. krusei exhibits considerable variation in its sensitivity to amphotericin B. The data presented in this paper (Table 1) are consistent with this observation. The sterols extracted from each strain are shown in Table 2. All resistant strains have substantially lower sterol content than their wild-type sensitive counterparts. ~ r o - mtheir chromatog~aphic mobilities, the unknown sterol at Rc 99/20 may possibly be a tetraene ergosterol precursor and those at Rc 91/20 and 105120, four monomethyl triterpenes. Previous reports on C. albicans ( 5 ) , Saccl~aronlyces cereuisiae (2), Neurospora crassa (18), Aspergillus fenneliae (14), and Cryptococcus neoforn~ans (13) indicate that the mode of polyene resistance is associated with lesions in the sterol pathway resulting in accumulations of some intermediate that has a polyene affinity lower than the wild-type end product sterol (usually ergosterol). The total sterol concentrations of the mutant-resistant strains were about the same as those of their wild-type sensitive progenitors. If sterols are the significant factors involved with polyene sensitiCity then it is evident from the data presented in this paper that TCH-T2672, TCH-T 1 12 1, and TCH-S 1 163 achieve polyene resistance simply by producing less sterol. Less sterol, and in particula; less ergosterol in the membrane of resistant strains would result in fewer polyene-sterol binding sites which are responsible for the pore formation through which cytoplasmic leakage occurs
(4). Some of the minor sterols are probably sterol intermediates with lower polyene affinity (12) and able to substitute in the physiological role played by ergosterol. However, such resistance mechanisms cannot be totally resolved till the function and polyene affinities for the esterified (16) and polymannan complexed (20) ergosterol in the membrane and cell wall is known.
Acknowledgment The advice and guidance of Dr. P. Fleming and technical assistance of Miss A. Robson of the Department of Bacteriology, Toronto Children's Hospital, is gratefully acknowledged. This research was supported by the University of Guelph Research Advisory Board Grant GR23 and the National Research Council Grant A6308. I. ATHAR,M . A,, and H . I. W I N N E R1971. . The development of resistance by Crrrrrlirlrr species to polyene antibiotics ;/I t~itro.J. Med. Microbial. 4: 505-5 17. D. H. R., J. E. T. C O R R I ED., A. WIDDOW2. BARTON, SON, M. BARK, and R. A. WOODS.1974. Biosynthesis of terpenes and steroids, part IX: the sterols of some mutant yeast and their relationship to the biosynthesis of ergosterol. J. Chem. Soc. Perkin, I: 13261333. 3. BRYSON,V., and W. S Z Y B A L S K1952. ~ . ~Microbiol selections. Science, 116: 45-52. A,, and A. CASS. 1968. Permeability 4. FINKELSTEIN, and electrical properties of thin layer membranes. J. Gen. Physiol. 52: 145-173. M., A. C. OEHLSCHLAGER, and A. M. U N 5. FRYBERG, RAU.1976. Sterol biosynthesis in antibiotic sensitive and resistant Carididrr. Arch. Biochem. Biophys. (In press.) 6. HAMILTON-MILLER, J. M. T. 1972. Physiological properties of mutagen-induced variants of Crrr~rlido
SAFE ET AL.
trlbictrris resistant to polyene antibiotics. Med. Mi-
7.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
8. 9. 10. 11.
12.
13.
14.
15.
crob~ol.Immunol. 5: 425-440. HAMILTON-MILLER. M. J. T. 1972. A comparaflve 111 1,itro study of amphotericin B clotrimazole and 5-fluorocytosine against clinically isolated yeasts. Sabouraudla, 10: 276-283. HAMILTON-MILLER, J. M. T 1972. Sterols from polyene-resistant mutants of Corrclitlrr rrll>itrrti\. J. Gen. Microblol. 73: 201-203. H A M I L T O N - M I LJ.L M. ~ RT. . 1974 Non-emergence of polyene-resistant yeasts: an hypothesis. Microbios, 10A: 91-95. E. K., and M. SOLOTOROVSKY. 1962. DeHEBEKA, velopment of strains of Corirlido rrll>rc(rri\ resistant candidin. J. Bactenol. 84: 237-241. HEBEKA, E. K., and M. SOLOTOROVSKY. 1965. Development of resistance to polyene antibiotics in Ctrrlrlrclo trlhic~rrrs.J . Bacteriol. 89: 1533-1539. JOHNSON, D., and R. SUBDEN. 1977. Polyene antlb~otic affinities for the sterols of resistant and sensitive stralns of Ncrrr.o~por.rrc.r.tr.\scr. Can. J. Mlcrobiol. 23: 113-1 15. KIM.S. J . , and K. J. KWON-CHUNG. 1974. Polyene resistant mutants ofA~pergillrrsferirrellitrr. Sterol contents and genetics. Antlmicrob. Agents Chemother. 6: 120-1 13. W. A. M I L N EW. . K I M S. . J . , K. J. KWON-CHUNG.G. B. H I L L and , G. PATTERSON 1975. Relatlonshlp between polyene resistance and sterol compositions in Cr?.ptot.ot t 11s I I P ~ ~ ~ I . I ? I ( IAntlmicrob. IIJ. Agents Chemother. 7: 99-106. LITTMAN, M. L., M. A. PISANO, and R. M. LANCAS-
16.
17. 18.
19.
20.
21. 22.
40 1
TER. 1957. Induced resistance of Ctrritlidrr species to nystatin and amphotericin B. Antibiot. Annu. 58: 98 1-987. MADYASTHA, P. B., and L. W. PARKS.1969. The effects of culture conditions on the ergosterol ester components of yeast. Biochim. Biophys. Acta, 176: 858462. MATHESON, K. 1974. The differentiation of C(rrrr1itlrr oll>ic~rrrsfrom other yeast and yeast-like organisms. Can. J. Med. Technol. 36: 93-118. MORRIS.D. C., S. SAFE,and R. E. S U B D E N1974. . Detection of the ergosterol and episterol isomers lichesterol and fecosterol in nystatin-resistant mutants of Nrrrr~ospo,vrcr.ossrr. Biochem. Genet. 12: 459466. SORENSON. L. J., E. G. MCNALL, and T. H. STERNBERG. 1958. The development of strains of C(r11rlit1troll~ic.orrsand Coc~c.idiotl~s ir?ir?liri.c-which are resistant to amphotericin B. Antibiot. Annu. 59: 920-923. THOMPSON, E. D., B. A. KNIGHTS. and L . W. PARKS. 1973. Identification and properties of a sterol-binding polysacchalide isolated from Sot~t~lrrrr~on~~cc.s cere~~isicrt,. Biochim. Biophys. Acta,304: 132-141. WOODS.R. A. 1971. Nystatin resistant mutants of yeast: alternations in sterol content. J. Bacteriol. 69-73. WOODS.R. A,, M. BARD.1. E. JACKSON, and D. J. DRUTZ.1974. Resistance to polyene antibiotics and correlated sterol changes in two isolates of Corrtlitltr rr~opic~olisfrom a patient with an amphotericin B-resistant funguria. J. Infect. Dis. 129: 53-58.
D L J P ( I ~ / I ?(?f I(C , I /I I/ L ~ I ? I ~U~li~.ersi/?. J/I?, o f G ~ ~ ~ , lGrrelpll, ph, 011/., C O I I ( I ~ ~ I AND
R. E . SUBDEN A N D D. C. MORRIS Depot~tt?~etlt of G e r ~ e t i cU/li~.ersit.v ~, of G~relph,Grtelpl~,Orlt., Cnrlnrlo Accepted December 20, 1976
SAFE.L. M., S. H. SAFE,R. E. SUBDEN, and D. C. MORRIS.1977. Sterol content and polyene Cotldidrl pcrroLrlrsri, and Ctrtlditlrr antibiotic resistance in isolates of Co~ididaXr~~sei. /ropic~olis.Can. J . Microbiol. 23: 39W01. Three isolates, one from each species of Corididl~L-r~rsei,C . pnrokrirsei, and C . tropicolis, obtained from infected patients, were more tolerant of significantly higher concentrations of polyene antibiotics than the corresponding reference wild types. The resistant strains isolated had the same sterols as their wild-type counterparts but in lowerconcentrations. SAFE,L. M., S. H. SAFE,R. E. SUBDEN et D. C. MORRIS.1977. Sterol content and polyene antibiotic resistance in isolates of Candida knrsei, Candida parakrrrsei, and Candidn rropicalis. Can. J . Microbiol. 23: 398-401. Trois isolats, un de chaque espece de Candida krrrsei, C. parakrirsei et C. tropicalis, isolCs de patients infect&, tolerent de f a ~ o nsignificative des concentrations plus elevkes d'antibiotiques polyenes que les souches sauvages correspondantes utilisees comme timoins. Les .ouches resistantes isolees ont les memes stCrols que les types sauvages, mais en concentrations plus faibles. [Traduit par le journal]
Introduction I t has been previously reported that Candida can he routinely mutated to polyene antibiotic resistance under laboratory conditions (1, 6). These mutant strains may be either single gene mutations (8, 21) involving discrete lesions in the sterol biosynthetic pathway (2, 18) or multistep variants from training experiments using serial transfers through solid media containing increasing concentrations of the antibiotic (10, 11, 15, 19). Although polyene antibiotics have been used therapeutically for 15 years, reports of clinically isolated polyene-resistant strains of Candida are surprisingly few (9, 22). For some reason there has been n o selection for antibiotic-resistant Candida under present clinical procedures. In this study, 864 isolates of Candida, obtained from patients at the Toronto Children's Hospital, were given a preliminary screening for polyene sensitivity on Szybalski (3) plates. Three polyeneresistant strains were isolated. Taxonomic studies indicated that the resistant strains were C. krusei, C. parakrusei, and C. tropicalis. This
paper reports on the sterol content of the resistant strains, reference wild-type or sensitive strains, and then suggests a possible correlation between the sterol content and polyene resistance.
Materials and Methods Strains and Crrlture Cot~rlitiot~s The wild-type C. olbicans ATCC 24433 was obtained from the American Type Culture Collection. Reference wild-type cultures of C. krrrsei, C . porakrrwei, and C. tropicolis were obtained from D r . P. Fleming, Department of Bacteriology, Toronto Children's Hospital (TCH). Resistant strain C. krrrsei TCH-T2672 was isolated from the urine of a 17-month-old male admitted for correction of a bladder extrophy. The patient had received nystatin treatment G times before the isolation of this strain. Cntldido parokrrrsei TCH-T1 1 21 was isolated from the urine of a 9-month-old female admitted for gastroenteritis and otitis media. She had received ampicillin and trimethoprim-sulphamethaoxzole but no polyene antibiotic. Cottdirla tropicalis TCH-S1163 was isolated from the stool of a 10-year-old male admitted for idiopathic acquired pancytopenia. He had received nystatin treatment for 4 d before isolation of this strain. Unless otherwise specified, wild-type cultures were kept on yeast propagation medium (YPM) consisting of 0.3% malt extract, 0.3% yeast extract, 0.5% peptone,
SAFE E T AL.
TABLE1. Minimum inhibitory concentrations of amphotericin B, candicidin, nystatin, and pimaricin for wild-type and resistant strains of C. krusei, C. parakrrrsei, C. fropicalis, and C. albicarls -
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
Strain
Candida krlcsei (wild type) C. krusei-R (isolate TCH-T2672) C. parakrusei (wild type) C. parakr~rsei-R (isolate TCH-TI 121) C. tropicalis (wild type) C. tropicalis-R (isolate TCH-S1163) C , albicans (wild type ATCC 24433)
Amphotericin B, ~g/ml
-
Candicidin, Nystatin, ~g/ml dm1
-
Pimaricin, ~g/ml
0.6
0.4
4.0
3.5
70.0
3.0
15.0
5.0
1.5
0.5
4.0
3.0
20.0
2.0
5.0
5.0
2.0
0.8
8 .O
5.0
25.0
2.0
10.0
5.0
0.5
0.3
3.0
3.5
1.0% glucose, and where necessary 1.5% agar. Resistant strains were kept on YPM plus 10 ug/ml nystatin.
Polyetle Antibiotics Antibiotics were stored at - 17°C till immediately before use. Stock solutions of amphotericin B (1 mg/ml), candicidin (1 mg/ml), and nystatin (10 mg/ml) were prepared in dimethylsulphoxide (DMSO). Piniaricin (I mg/ml) was dissolved in propylene glycol. Freshly prepared solutions of antibiotics were added to the medium after it had cooled to 60°C after autoclaving. Sensitivity Tests As a preliminary screening procedure, 4 of the 864 strains isolated plus a wild-type control (C. nlbicnrls ATTC 24433) were streaked on each plate. A suspension of each strain was made in 0.14 M saline One loopful of each suspension was streaked on a Szybalski (3) gradient plate at 200 ~ g / m nystatin. l Square (100 x 15 mni) petri plates were used for all tests. Cultures were incubated at 37'C and growth scored after 48 h. The minimum inhibitory concentrations (MIC) were determined by plate-dilution tests. At first the antibiotics l were incorporated Into the medium at 0.5 ~ g / mincrements from 0.0 to 100.0 g/ml. Succeeding experiments used 0.02 ~ g / m lincrements over a narrower range. Aliquots containing 0.1 ml of 2 x l o 5 cells/ml in 0.14 M sallne were spread uniformly on plates containing the antibiotics. Sepnrafion ntld Idetltificatiot~of Stet 01s Forty-eight hour, air-agitated (bubblers), 3-! cultures of Cnndida were harvested by centrifugation, washed twice with 0.14 M saline, and lyophilized. The cells were then macerated and refluxed in 300 ml of 10% K O H in methanol for 2 h. Cell wall fragments and other debris were removed by filtrat~onthen washed with ether. The filtrate was diluted with 2 volumes of water and extracted with 2 volunies of diethyl ether. The ether phase was washed wlth water, dried over Na,SO,, concentrated to dryness in a flash evaporator, resuspended in a small volume of benzene, and applied to a preparative thinlayer plate (silica gel H F 254 + 366) then developed with
hexane:ether:ethanol (80:20:2). Sterols detected by UV quenching were removed from the plate, eluted from the silica gel, and converted into their corresponding acetate derivatives with acetic anhydride pyridine (2: 1). Steryl acetates were purified by preparative thin-layer chromatography as described above and analyzed on HewlettPackard 700 and Beckman GC72 gas chronlatographs using 8 ft x & in. glass columns packed with 1.5% OV17 on Gas Chrom. Q at 2605C. The carrier gas was helium at 60 ml/min and the detector and inlet temperatures were 290°C. Internal standards for sterol identification were obtained from a previous study (18).
Results and Discussion Of 864 isolates tentatively identified as Ccrtidiu'u and screened for polyene sensitivity, three demonstrated unusual resistauce to polyene antibiotics on Szybalski plate gradients. Further taxonomic tests including germ-tube formation, fermentation reactions, and growth on corn meal Tween 80 media according to Matheson (17) indicated that strain TCH-T2672 was C. tropicalis, TCH-TI 12 1 was C. paraknrsei, and TCHS1163 was C. knrsei. Polyene MIC for each of these isolated strains were compared to their wild types as shown in Table 1. The data indicate that the wild-type strains were more sensitive to all of the antibiotics used and that candicidin was more potent than pimaricin. It should also be noted that the difference in MIC for all resistant strains was greatest for amphotericin, and least for pirnaricin. In fact, C. tropicnlis, TCH-S 1 163, and wild-type C. tropicalis had identical MIC's for pirnaricin. In a previous report, Hamilton-Miller (7) identified two strains
CAN. J.
MICROBIOL. VOL.
23, 1977
TABLE2. Sterol content of various Candida strains expressed as pg sterol/mg dry weight
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/14/14 For personal use only.
Candida krusei
Candida parakrusei
Candida fropicalis
Sterol*
Rct
Wild type
Resistant (TCH-T2672)
Wild type
Resistant (TCH-TI 121)
Wild type
Lichesterol Ergosterol Fecosterol Unknown sterols
77/20 82/20 91/20 99/20 91/20 l05/20
53 .O 210.0 320.0 -
6.0 4.0 2.0 9.0
62.0 920.0 20.0
89.0 360.0 14.0 36.0 -
11 . O 50.0 1.1 -
-
1 .O 28.0 1 .O 5.6 0.6 2.0
583.0
21 .O
1002.0
38.2
499.0
62.1
-
Resistant (TCH-S1163)
'Identified by comparison with authentic standards (16). tRetention time on gas chromatographs 1.5% OV-17, 260°C, relative to internal standard cholestane.
of C. krusei out of 413 clinically isolated yeasts. One had an MIC of 2 pg/ml and the other 16 pg/ml. Further work with other strains showed that C. krusei exhibits considerable variation in its sensitivity to amphotericin B. The data presented in this paper (Table 1) are consistent with this observation. The sterols extracted from each strain are shown in Table 2. All resistant strains have substantially lower sterol content than their wild-type sensitive counterparts. ~ r o - mtheir chromatog~aphic mobilities, the unknown sterol at Rc 99/20 may possibly be a tetraene ergosterol precursor and those at Rc 91/20 and 105120, four monomethyl triterpenes. Previous reports on C. albicans ( 5 ) , Saccl~aronlyces cereuisiae (2), Neurospora crassa (18), Aspergillus fenneliae (14), and Cryptococcus neoforn~ans (13) indicate that the mode of polyene resistance is associated with lesions in the sterol pathway resulting in accumulations of some intermediate that has a polyene affinity lower than the wild-type end product sterol (usually ergosterol). The total sterol concentrations of the mutant-resistant strains were about the same as those of their wild-type sensitive progenitors. If sterols are the significant factors involved with polyene sensitiCity then it is evident from the data presented in this paper that TCH-T2672, TCH-T 1 12 1, and TCH-S 1 163 achieve polyene resistance simply by producing less sterol. Less sterol, and in particula; less ergosterol in the membrane of resistant strains would result in fewer polyene-sterol binding sites which are responsible for the pore formation through which cytoplasmic leakage occurs
(4). Some of the minor sterols are probably sterol intermediates with lower polyene affinity (12) and able to substitute in the physiological role played by ergosterol. However, such resistance mechanisms cannot be totally resolved till the function and polyene affinities for the esterified (16) and polymannan complexed (20) ergosterol in the membrane and cell wall is known.
Acknowledgment The advice and guidance of Dr. P. Fleming and technical assistance of Miss A. Robson of the Department of Bacteriology, Toronto Children's Hospital, is gratefully acknowledged. This research was supported by the University of Guelph Research Advisory Board Grant GR23 and the National Research Council Grant A6308. I. ATHAR,M . A,, and H . I. W I N N E R1971. . The development of resistance by Crrrrrlirlrr species to polyene antibiotics ;/I t~itro.J. Med. Microbial. 4: 505-5 17. D. H. R., J. E. T. C O R R I ED., A. WIDDOW2. BARTON, SON, M. BARK, and R. A. WOODS.1974. Biosynthesis of terpenes and steroids, part IX: the sterols of some mutant yeast and their relationship to the biosynthesis of ergosterol. J. Chem. Soc. Perkin, I: 13261333. 3. BRYSON,V., and W. S Z Y B A L S K1952. ~ . ~Microbiol selections. Science, 116: 45-52. A,, and A. CASS. 1968. Permeability 4. FINKELSTEIN, and electrical properties of thin layer membranes. J. Gen. Physiol. 52: 145-173. M., A. C. OEHLSCHLAGER, and A. M. U N 5. FRYBERG, RAU.1976. Sterol biosynthesis in antibiotic sensitive and resistant Carididrr. Arch. Biochem. Biophys. (In press.) 6. HAMILTON-MILLER, J. M. T. 1972. Physiological properties of mutagen-induced variants of Crrr~rlido
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