Regístrese
Buscar en
Revista Iberoamericana de Micología
Toda la web
Inicio Revista Iberoamericana de Micología In vitro antifungal susceptibility pattern and ergosterol content in clinical ye...
Información de la revista
Vol. 28. Núm. 2.
Páginas 100-103 (Abril - Junio 2011)
Compartir
Compartir
Descargar PDF
Más opciones de artículo
Visitas
3795
Vol. 28. Núm. 2.
Páginas 100-103 (Abril - Junio 2011)
Note
DOI: 10.1016/j.riam.2010.12.003
Acceso a texto completo
In vitro antifungal susceptibility pattern and ergosterol content in clinical yeast strains
Perfil de sensibilidad in vitro y contenido en ergosterol en levaduras de origen clínico
Visitas
3795
Alicia Gomez-Lopez??
Autor para correspondencia
aliciagl@isciii.es

Corresponding author.
, María Jose Buitrago, Juan Luis. Rodriguez-Tudela, Manuel Cuenca-Estrella
Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, España
Este artículo ha recibido
3795
Visitas
Información del artículo
Resumen
Texto completo
Bibliografía
Descargar PDF
Estadísticas
Tablas (1)
Table 1. Ergosterol content (means of triplicate) and antifungal susceptibility profile of strains included in the study.
Abstract
Background

Absence or severe reduction in the amount of ergosterol in the fungal membrane and its replacement with other sterols have been described as potential antifungal resistance mechanisms in fungi.

Aims and methods

The ergosterol content in a collection of 51 clinical yeast isolates, including susceptible and resistant strains to amphotericin B and azoles, was estimated by a simple chromatographic method (HPLC-UV).

Results

A high content of ergosterol was detected for several strains of Candida glabrata, Candida tropicalis or Pichia membranifaciens. In contrast, strains of Cryptococcus neoformans and Dipodascus capitatus had the lowest ergosterol concentrations. No significant correlation was observed between antifungal susceptibility patterns and ergosterol content.

Conclusions

We conclude from this study that ergosterol content on yeasts may not be associated with specific resistant patterns.

Keywords:
HPLC-UV
Resistance mechanisms
Yeasts
Resumen
Antecedentes

La ausencia o disminución en la cantidad de ergosterol, así como su sustitución por otros esteroles en la membrana, se ha considerado como un posible mecanismo de resistencia de la célula fúngica.

Objetivos y métodos

En este trabajo hemos evaluado la cantidad de ergosterol de una colección de 51 aislamientos clínicos de levaduras, incluyendo cepas sensibles y resistentes a antifúngicos, mediante un sencillo método cromatográfico (HPLC-UV).

Resultados

Algunas cepas de Candida glabrata, Candida tropicalis y Pichia membranifaciens mostraron mayor contenido en ergosterol que el resto, mientras que las de Cryptococcus neoformans y Dipodascus capitatus presentaron el contenido más bajo. No se observó ninguna relación con suficiente potencia estadística entre el patrón de sensibilidad in vitro y el contenido de ergosterol.

Conclusiones

Podemos concluir de este estudio que el contenido en ergosterol no se relaciona sistemáticamente con un patrón de resistencia definido.

Palabras clave:
HPLC-UV
Mecanismos de resistencia
Levaduras
Texto completo

Ergosterol is a specific lipid of fungal membranes,22 absent in plants and animals, widely used as an index of fungal mass, particularly in the food-agriculture industry.9,11,18 This special quality has also been exploited by pharmaceutical research as a specific target for antifungal drug development, with this sterol being the focus of many reports in the literature. The mechanisms of action of polyenes and azoles involve ergosterol. Polyenes appear to interact with sterols directly, inducing formation of aqueous pores in lipid bilayers and leading to cell death, whereas azoles inhibit the ergosterol biosynthetic pathway, leading to inhibition of cell growth and replication.5 Both classes of antifungal compounds remain among the most effective available agents in the clinics. Absence, severe reduction, high amount or even ergosterol replacement with other sterols have all been considered as potential mechanisms of resistance against both antifungal classes in fungi.8,13,19,20,21 The fact that several studies on resistant mutant strains have shown changes in sterol pattern suggests a connection between ergosterol content and antifungal drug resistance.24,25 We describe here a simple chromatographic method to analyze ergosterol from yeast cells in order to investigate its potential association with antifungal drug susceptibility patterns.

A total of 51 clinical yeast strains (from 15 different species) were studied, including strains with resistance or decreased susceptibility in vitro to amphotericin B (AMB) and azoles. Isolates were identified by standard mycological methods.7 Two ATCC strains (C. krusei ATCC 6258 and C. parapsilosis ATCC 22019) were included in order to confirm the reproducibility of results.

For susceptibility testing we followed strictly the recommendations proposed by EUCAST.14 Minor modifications were included to improve the growth of Cryptococcus neoformans strains and other species showing poor growth (Rhodotorula spp., Dipodascus spp.).6 Antifungal agents tested were AMB (16-0.03μg/ml); fluconazole, FLC (64-0.125μg/ml); itraconazole, ITC (8-0.015μg/ml); and voriconazole, VRC (8-0.015μg/ml).

Strains were classified as resistant or susceptible taking into account interpretative breakpoints proposed by EUCAST for FLC and VRC,15,16 or CLSI for the remaining agents.12 Of note, AMB breakpoints are not available yet, but clinical resistance has been associated with MIC>1μg/ml.4

Yeast cells were cultured in 10ml of RPMI 1640 medium-2% glucose at 35°C for 48h (2.5×105 cells/ml). Cells were then harvested by centrifugation at 2,500 x g for 5min. The resultant pellet was washed and weighed out after removing all visible water (wet weight). Total sterols were extracted from each pellet as reported previously.1 Prior to chromatographic analysis, each dry residue was dissolved in 1ml of methanol, filtered to remove any particle in suspension, and finally aliquots of 50μl were injected into the HPLC system (LC Module I HPLC Waters). Loss of ergosterol was not detected after the filtration process (data not shown). A flow rate of 1ml/min (methanol 100%) was established, and a detector wavelength of 282nm was selected for quantification purposes. Six solutions of known concentrations of ergosterol (from 1 to 50μg/ml) were also injected into the system to serve as reference for qualitative and quantitative analysis (Standard Curve). These solutions were prepared diluting purified ergosterol powder (Fluka, Madrid, Spain) in methanol. (It is recommended to wait around 10minutes for its complete solubilization). Concentration values, expressed as μg of ergosterol per ml of solvent (methanol), were determined by the wet weight (mg) and ultimately expressed as μg/mg. To test extraction efficiency several experiments were performed by comparing results from samples spiked with known amounts of ergosterol. Experiments were repeated up three times. Moreover, cultures of ATCC strains were also tested repeatedly in order to evaluate the robustness of the method.

Ergosterol was detected and characterized approximately 7.5±0.3min after injection. The standard curve was linear over a concentration range between 1 and 50μg/ml of ergosterol (r2=0.991). The assay showed an acceptable reproducibility, with variation coefficients lower than 10%. The lower limit of detection was established at 1μg/ml. The recovery rate from spiked samples was in the range of 72-76%.

For ATCC strains acceptable variability in results was found (0.21±0.04μg/mg and 0.14±0.04μg/mg for C. krusei and C. parapsilosis respectively after five independent experiments).

Table 1 summarizes the ergosterol amounts for all yeast strains included in the study. The average ergosterol content was 0.16 (ranging from not detected to 0.43) μg/mg of weight. The wet weight ranged between 28.1 and 151.3mg.

Table 1.

Ergosterol content (means of triplicate) and antifungal susceptibility profile of strains included in the study.

Strain (n)  Ergosterol/wet weight μg/mg  MIC values (μg/ml)
    AMB  FLC  ITC  VRC 
Candida albicans (7)  0.11 (0.07-0.21)         
CL-3700  0.09  0.12  64 
CL-3734  0.21  0.5  64 
CL-3814  0.08  0.06  64  >8  >8 
CL-3464  0.07  0.03  0.12  0.015  0.015 
CL-3477  0.11  0.06  0.12  0.015  0.015 
CL-3433  0.07  0.03  0.12  0.015  0.015 
CL-3485  0.13  0.06  0.12  0.015  0.015 
Candida glabrata (3)  0.29 (0.26-0.32)         
CL-3465  0.26  0.06  0.5  0.5 
CL-3471  0.32  0.12  16  0.5  0.5 
CL-3447  0.29  0.12  0.5  0.5 
Candida parapsilosis (4)  0.14 (ND-0.25)         
CL-3472  0.25  0.06  0.12  0.015  0.015 
CL-3478  ND  0.25  0.25  0.03  0.015 
CL-3473  0.19  0.12  0.5  0.015  0.015 
Cp ATCC 22019  0.14  0.25  0.06  0.015 
Candida krusei (3)  0.22 (0.19-0.26)         
CL-3476  0.26  0.25  32  0.25  0.5 
CL-3453  0.19  0.25  32  0.25  0.5 
Ck ATCC6258  0.21  0.25  32  0.06  0.12 
Candida tropicalis (6)  0.23 (ND-0.43)         
CL-2815  0.35  0.25  0.12  0.015  0.015 
CL-3780  0.22  0.06  0.12  0.015  0.015 
CL-3285  0.11  0.5  32  0.5  0.5 
CL-4019  0.29  64  > 8  > 8 
CL-4021  0.43  64  > 8  > 8 
CL-3730  ND  64  > 8  > 8 
Candida guilliermondii (3)  0.13 (0.08-0.19)         
CL-1904  0.19  64 
CL-2130  0.08  0.5  32  0.5  0.25 
CL-3301  0.14  0.5  32  0.5  0.25 
Candida pelliculosa (1)  0.22         
CL-3769  0.22  0.06  16  0.5  0.25 
Candida lusitaniae (9)  0.19 (0.10-0.25)         
CL-523  0.23  0.03  0.12  0.015  0.015 
CL-524  0.22  0.12  0.5  0.015  0.015 
CL-1039  0.23  0.03  0.12  0.015  0.015 
CL-841  0.25  0.03  0.25  0.03  0.015 
CL-1508  0.12  0.03  0.5  0.03  0.015 
CL-2518  0.17  0.03  0.12  0.015  0.03 
CL-3719  0.21  0.03  0.5  0.03  0.015 
CL-2602  0.15  0.03  0.25  0.015  0.015 
CL-3927  0.10  64  0.12  0.5 
Candida famata (2)  0.14 (0.14-0.14)         
CL-3728  0.14  16  0.25 
CL-3756  0.14  0.25  32  0.5 
Saccharomyces cerevisiae (1)  0.21         
CL-3030  0.21  32  0.5  0.5 
Pichia membranifaciens (2)  0.28 (0.26-0.30)         
CL-3360  0.26  0.5  64  0.5  0.25 
CL-3679  0.30  0.12  64  0.25  0.25 
Candida haemulonii (1)  0.10         
CL-3458  0.10  64  0.12  0.12 
Cryptococcus neoformans (3)  0.07 (0.06-0.07)         
CL-2620  0.06  64  >8  >8 
CL-3152  0.07  0.12  16  0.25  0.12 
CL-3789  0.07  0.5  16  0.5  0.12 
Rhodotorula mucilaginosa (3)  0.11 (0.06-0.15)         
CL-2358  0.06  64 
CL-2401  0.15  64 
CL-2544  0.14  64 
Dipodascus capitatus (3)  0.06 (0.02-0.13)         
CL-3500  0.02  32  0.5  0.25 
CL-3913  0.13  0.5  32  0.25  0.5 
CL-3914  0.04  0.5  32  0.25  0.25 
Total (51)  0.16 (0.00-0.43)         

ND: not detected; ergosterol content were under the limit of quantification defined for the method described in the text. $Clinical resistance has been associated with AMB MIC>1, FLC>2, ITC>0.125 and VRC>0.125 μg/ml (see text and references). In bold those MICs above breakpoints.

Several strains of C. glabrata (3 out of 3 included), C. tropicalis (3/6), Pichia membranifaciens (3/3) or C. krusei (1/3) showed a high content of ergosterol (> 0.25μg/mg). Conversely C. neoformans (3/3) or Dipodascus capitatus strains (2/3) had the lowest concentration (less than 0.07μg/mg). It is worth noting that C. neoformans is an encapsulated yeast, so its ergosterol content may seem lower when correlated to wet weight. However, the low number of representative strains of each species included in the study made it difficult to detect significant differences between species.

The antifungal susceptibility profile is also summarized in Table 1. Results do not show any direct correlation between MICs and ergosterol levels for any of the antifungal agents tested. Calculated Spearman Rho coefficients (SPSS, version 17.0, Madrid, Spain) were as follows: –0.201, –0.126, –0.107 and –0.039 for AMB, FLC, ITC and VRC respectively (p>0.05). For strains showing AMB MICs higher than 1μg/ml a great variability in ergosterol content was detected (Table 1) and differences with AMB susceptible strains were not statistically significant. In the same way, no differences were noted between FLC, ITC or VRC resistant and susceptible strains studied.

Results described here show no significant correlation between MICs and ergosterol content. In fact the ergosterol content estimated in such an heterogeneous collection of yeasts revealed a great degree of variability, even for members belonging to the same species or those included in the same category (resistant or susceptible to a given antifungal). It is worth noting that the few representative strains of some categories (only 4 AMB resistant strains) limit any conclusions that can be drawn. However, several interesting observations can be made. Our results support those recently described by other authors reporting that several mechanisms might contribute to a step-by-step acquisition of resistance in yeasts17 and also, to a variable ergosterol composition.3,19,20,21 In fact, the susceptibility profile may not depend on the membrane sterol content alone. Other constituents of the cell, and especially the cell wall, might contribute to resistance.10 Therefore, only membrane sterol content does not enable us to explain resistance or susceptibility of yeasts to AMB or azoles, since resistant strains could be as rich in ergosterol as susceptible strains,2,21 as it has also been described here. It is unquestionable that molecular characterization of mechanisms for the acquisition of resistance in these strains might clarify the real role of ergosterol in antifungal resistance. In our opinion this is a limitation of this study, so further molecular approaches are warranted to better characterize the role of ergosterol content in yeast resistance.

To our knowledge, this is the first report correlating ergosterol content and antifungal susceptibility in a collection of susceptible and resistant strains using a chromatographic method (HPLC-UV). This HPLC assay has proved to be a useful method for ergosterol characterization and quantification, even superior to those that use sole ultraviolet absorption spectroscopy.23 However, taken into account the multifactorial nature of the resistance in yeasts, any change in the ergosterol content may not be directly associated with a defined pattern of resistance, although it might contribute to the prediction of possible mechanisms. Further molecular studies are needed to fully determine and characterize resistance mechanism(s) in these fungal cells

Conflicts of interest

In the past 5 years, M.C.E. has received grant support from Astellas Pharma, bioMérieux, Gilead Sciences, Merck Sharp and Dohme, Pfizer, Schering Plough, Soria Melguizo SA, the European Union, the ALBAN program, the Spanish Agency for International Cooperation, the Spanish Ministry of Culture and Education, the Spanish Health Research Fund, the Instituto de Salud Carlos III, the Ramon Areces Foundation, the Mutua Madrileña Foundation. He has been an advisor/consultant to the Panamerican Health Organization, Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Schering Plough. He has been a paid guest speaker on behalf of Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Schering Plough.

In the past 5 years, J.L.R.T. has received grant support from Astellas Pharma, Gilead Sciences, Merck Sharp and Dohme, Pfizer, Schering Plough, Soria Melguizo SA, the European Union, the Spanish Agency for International Cooperation, the Spanish Ministry of Culture and Education, the Spanish Health Research Fund, the Instituto de Salud Carlos III, the Ramon Areces Foundation, the Mutua Madrileña Foundation. He has been an advisor/consultant to the Panamerican Health Organization, Gilead Sciences, Merck Sharp and Dohme, Mycognostica, Pfizer, and Schering Plough. He has been a paid guest speaker on behalf of Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Schering Plough.

Other authors: No conflicts

References
[1]
B.A. Arthington-Skaggs, W. Lee-Yang, M.A. Ciblak, J.P. Frade, M.E. Brandt, R.A. Hajjeh, et al.
Comparison of visual and spectrophotometric methods of broth microdilution MIC end point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of fluconazole and itraconazole against trailing and nontrailing Candida isolates.
Antimicrob Agents Chemother., 46 (2002), pp. 2477-2481
[2]
K. Bahmed, R. Bonaly, J. Coulon.
Relation between cell wall chitin content and susceptibility to amphotericin B in Kluyveromyces, Candida and Schizosaccharomyces species.
Res Microbiol., 154 (2003), pp. 215-222
[3]
S. Brun, T. Berges, P. Poupard, C. Vauzelle-Moreau, G. Renier, D. Chabasse, et al.
Mechanisms of azole resistance in petite mutants of Candida glabrata.
Antimicrob Agents Chemother., 48 (2004), pp. 1788-1796
[4]
M. Cuenca-Estrella, D. Rodriguez, B. Almirante, J. Morgan, A.M. Planes, M. Almela, et al.
In vitro susceptibilities of bloodstream isolates of Candida species to six antifungal agents: results from a population-based active surveillance programme, Barcelona, Spain, 2002–2003.
J Antimicrob Chemother., 55 (2005), pp. 194-199
[5]
A. Espinel-Ingroff.
Mechanisms of resistance to antifungal agents: yeasts and filamentous fungi.
Rev Iberoam Micol., 25 (2008), pp. 101-106
[6]
A. Gomez-Lopez, E. Mellado, J.L. Rodriguez-Tudela, M. Cuenca-Estrella.
Susceptibility profile of 29 clinical isolates of Rhodotorula spp. and literature review.
J Antimicrob Chemother., 55 (2005), pp. 312-316
[7]
C.P. Kurtzman, J.W. Fell.
The Yeasts. A Taxonomic Study., 4th ed,
[8]
N.N. Mishra, T. Prasad, N. Sharma, A.A. Lattif, R. Prasad, D.K. Gupta.
Membrane fluidity and lipid composition in clinical isolates of Candida albicans isolated from AIDS/HIV patients.
Acta Microbiol Immunol Hung., 54 (2007), pp. 367-377
[9]
S.Y. Newell.
Estimating fungal biomass and productivity in decomposing litter.
The fungal community: its organization and role in the ecosystem, pp. 521-561
[10]
E. Ramanandraibe, M. Younsi, J. Coulon, V. Loppinet, A. Hakkou, R. Bonaly.
Implication of cell wall constituents in the sensitivity of Kluyveromyces lactis strains to amphotericin B.
Res Microbiol., 149 (1998), pp. 109-118
[11]
C. Ravelet, C. Grosset, J. Alary.
Quantitation of ergosterol in river sediment by liquid chromatography.
J Chromatogr Sci., 39 (2001), pp. 239-242
[12]
Rex JH, Alexander BA, Andes D, Arthington-Skaggs B, Brown SD, Chaturvedi V, et al. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard-Third Edition. CLSI document M27-A3. 28-4-2008.
[13]
C.M. Richman-Boytas, L.W. Parks.
Effects of sterol alterations on nystatin sensitivity in Saccharomyces cerevisiae.
Microbios., 59 (1989), pp. 101-111
[14]
J.L. Rodriguez-Tudela, F. Barchiesi.
Subcommittee on Antifungal Susceptibility Testing (AFST). EUCAST Definitive Document EDef 7. 1: Method for the determination of broth dilution MICs of antifungal agents for fermentative yeast.
Clin Microbiol Infect., 14 (2008), pp. 398-405
[15]
J.L. Rodriguez-Tudela, J.P. Donnelly, M.C. Arendrup, S. Arikan, F. Barchiesi, J. Bille, et al.
EUCAST technical note on fluconazole.
Clin Microbiol Infect., 14 (2008), pp. 193-195
[16]
J.L. Rodriguez-Tudela, J.P. Donnelly, M.C. Arendrup, S. Arikan, F. Barchiesi, J. Bille, et al.
EUCAST Technical Note on voriconazole.
Clin Microbiol Infect., 14 (2008), pp. 985-987
[17]
D. Sanglard, F.C. Odds.
Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences.
Lancet Infect Dis., 2 (2002), pp. 73-85
[18]
L.M. Seitz, D.B. Sauer, R. Burroughs, H.E. Mohr, J.D. Dubbard.
Ergosterol as a measure of fungal growth.
Phytopathology., 69 (1979), pp. 1202-1203
[19]
P. Vandeputte, G. Larcher, T. Berges, G. Renier, D. Chabasse, J.P. Bouchara.
Mechanisms of azole resistance in a clinical isolate of Candida tropicalis.
Antimicrob Agents Chemother., 49 (2005), pp. 4608-4615
[20]
P. Vandeputte, G. Tronchin, G. Larcher, E. Ernoult, T. Berges, D. Chabasse, et al.
A nonsense mutation in the ERG6 gene leads to reduced susceptibility to polyenes in a clinical isolate of Candida glabrata.
Antimicrob Agents Chemother., 52 (2008), pp. 3701-3709
[21]
P. Vandeputte, G. Tronchin, F. Rocher, G. Reiner, T. Berges, D. Chabasse, et al.
Hypersusceptibility to Azole Antifungals in a Clinical Isolate of Candida glabrata with Reduced Aerobic Growth.
Antimicrob Agents Chemother., 53 (2009), pp. 3034-3041
[22]
J.D. Weete.
Structure and function of sterols in fungi.
Adv Lipid Res., 23 (1989), pp. 115-167
[23]
R.A. Woods, M. Bard, I.E. Jackson, D.J. Drutz.
Resistance to polyene antibiotics and correlated sterol changes in two isolates of Candida tropicalis from a patient with an amphotericin B-resistant funguria.
J Infect Dis., 129 (1974), pp. 53-58
[24]
L.Y. Young, C.M. Hull, J. Heitman.
Disruption of ergosterol biosynthesis confers resistance to amphotericin B in Candida lusitaniae.
Antimicrob Agents Chemother., 47 (2003), pp. 2717-2724
[25]
M. Younsi, E. Ramanandraibe, R. Bonaly, M. Donner, J. Coulon.
Amphotericin B resistance and membrane fluidity in Kluyveromyces lactis strains.
Antimicrob Agents Chemother., 44 (2000), pp. 1911-1916
Opciones de artículo
Herramientas
es en pt
Política de cookies Cookies policy Política de cookies
Utilizamos cookies propias y de terceros para mejorar nuestros servicios y mostrarle publicidad relacionada con sus preferencias mediante el análisis de sus hábitos de navegación. Si continua navegando, consideramos que acepta su uso. Puede cambiar la configuración u obtener más información aquí. To improve our services and products, we use "cookies" (own or third parties authorized) to show advertising related to client preferences through the analyses of navigation customer behavior. Continuing navigation will be considered as acceptance of this use. You can change the settings or obtain more information by clicking here. Utilizamos cookies próprios e de terceiros para melhorar nossos serviços e mostrar publicidade relacionada às suas preferências, analisando seus hábitos de navegação. Se continuar a navegar, consideramos que aceita o seu uso. Você pode alterar a configuração ou obter mais informações aqui.