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Comparison among tomato juice agar with other three media for differentiation of Candida dubliniensis from Candida albicans

Comparação do ágar suco de tomate com outros três meios, na diferenciação entre C. albicans e C. dubliniensis

Abstracts

The purpose of the present study is to compare the tomato juice agar, a well known medium employed to observe ascospore formation, with niger seed agar, casein agar and sunflower seed agar, applied to a differentiation between C. dubliniensis and C. albicans. After 48 hours of incubation at 30 ºC all 26 (100%) C. dubliniensis isolates tested produced chlamydospores on tomato juice agar as well as in the other three media evaluated. However, when we inoculated all media with C. albicans, the absence of chlamydospores became resulting in the following percents: tomato juice agar (92.47%), niger seed agar (96.7%), casein agar (91.39%), and sunflower seed agar (96.7%). These results indicate that tomato juice agar is another medium which can also be used in the first phenotypic differentiation between C. dubliniensis and C. albicans.

Candida dubliniensis; Tomato juice agar; Phenotypic identification


O presente estudo teve como objetivo comparar o ágar suco de tomate, um tradicional meio utilizado para observação de ascósporos em leveduras, com o ágar semente de niger, ágar caseína e ágar semente de girassol, na diferenciação fenotípica entre C. albicans e C. dubliniensis. Após 48 h de incubação a 30 ºC, os 26 isolados de C. dublinienis (100%) evidenciaram a formação de clamidoconídios igualmente em todos os meios comparados. Entretanto, quando semeados com C. albicans, a formação de clamidoconídios foi raramente observada, resultando nos seguintes percentuais de ausência destas estruturas: ágar suco de tomate (92,47%), ágar niger (96,7%), ágar caseína (91,39%), ágar semente de girassol (96,7%). Estes resultados permitem-nos sugerir a utilização do ágar suco de tomate como mais um meio que, já no primo-isolamento, é capaz de, presuntivamente, diferenciar C. albicans de C. dubliniensis.


MYCOLOGY

Comparison among tomato juice agar with other three media for differentiation of Candida dubliniensis from Candida albicans

Comparação do ágar suco de tomate com outros três meios, na diferenciação entre C. albicans e C. dubliniensis

Sydney Hartz AlvesI; Érico Silva de LoretoII; Carlos Eduardo LinaresII; Carolina P. SilveiraIII; Liliane A. ScheidIII; Daniela I. Brayer PereiraI; Janio Morais SantuarioI

IDepartamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria (UFSM). Santa Maria, RS, Brasil. Phone/fax (55) 3220-8906

IICurso de Pós graduação em Ciências Farmacêuticas, UFSM, Santa Maria, RS, Brasil

IIIAluno de Graduação em Farmácia e Bioquímica, PIBIC/CNPq.

Correspondence to Correspondence to: Sydney Hartz Alves Rua Andradas 1985/201 97010-033 Santa Maria, RS, Brasil e-mail: hartzsa@ccs.ufsm.br

SUMMARY

The purpose of the present study is to compare the tomato juice agar, a well known medium employed to observe ascospore formation, with niger seed agar, casein agar and sunflower seed agar, applied to a differentiation between C. dubliniensis and C. albicans. After 48 hours of incubation at 30 ºC all 26 (100%) C. dubliniensis isolates tested produced chlamydospores on tomato juice agar as well as in the other three media evaluated. However, when we inoculated all media with C. albicans, the absence of chlamydospores became resulting in the following percents: tomato juice agar (92.47%), niger seed agar (96.7%), casein agar (91.39%), and sunflower seed agar (96.7%). These results indicate that tomato juice agar is another medium which can also be used in the first phenotypic differentiation between C. dubliniensis and C. albicans.

Keyworrds:Candida dubliniensis; Tomato juice agar; Phenotypic identification.

RESUMO

O presente estudo teve como objetivo comparar o ágar suco de tomate, um tradicional meio utilizado para observação de ascósporos em leveduras, com o ágar semente de niger, ágar caseína e ágar semente de girassol, na diferenciação fenotípica entre C. albicans e C. dubliniensis. Após 48 h de incubação a 30 ºC, os 26 isolados de C. dublinienis (100%) evidenciaram a formação de clamidoconídios igualmente em todos os meios comparados. Entretanto, quando semeados com C. albicans, a formação de clamidoconídios foi raramente observada, resultando nos seguintes percentuais de ausência destas estruturas: ágar suco de tomate (92,47%), ágar niger (96,7%), ágar caseína (91,39%), ágar semente de girassol (96,7%). Estes resultados permitem-nos sugerir a utilização do ágar suco de tomate como mais um meio que, já no primo-isolamento, é capaz de, presuntivamente, diferenciar C. albicans de C. dubliniensis.

INTRODUCTION

Candida dubliniensis is a newly described fungus which was first reported by Sullivan et al. in 199521. C. dubliniensis is phylogenetically closely related to C. albicans, thereby sharing many morphological and physiological characteristics as germ tube positive, similar biochemical patterns and the ability to form chlamydospores in rice extract agar and cornmeal agar21,22.

Routine discrimination between C. dubliniensis and the closely related species C. albicans has been problematic21,22. The most accurate method of identifying C. dubliniensis and discriminating it from C. albicans requires PCR-based tests8,12,21,22; however, these are not readily applicable to the high-volume throughput of isolates in many diagnostic laboratories routine2,3,14.

The most reliable phenotypic methods for the identification of C. dubliniensis include carbohydrate assimilation profile analysis by using commercially available yeast identification systems5,7 and detection of differential antigen expression through immunofluorescence microscopy6. However, they can only be applied after the first isolation.

One of the key features employed in the initial description of C. dubliniensis was its ability to produce abundant chlamydospores on corn meal agar and rice-agar-Tween medium21. Based on this characteristic, recently new media as niger seed agar20, caffeic-acid-ferricitrate agar2, casein agar14, sunflower seed agar3 and tobacco agar10 have been proposed to differentiate both species simultaneously up to the first isolation.

In the last ten years there has been a proliferation of phenotypic tests in the literature for the differentiation of C. albicans and C. dubliniensis which includes carbohydrate assimilation profile5,7, appearance on CHROMagar11, ability to grow at 45 ºC16, inability to grow on Sabouraud dextrose broth with NaCl 6.5%4, absence of an opacity halo around an inoculated site on Tween 80 medium19 and some others6,15,18.

Tomato juice agar or V-8 juice agar is a well known medium widely used for ascospore formation in yeasts as Saccharomyces cerevisae and Hansenula anomala1,9,13.

Here we compare the tomato juice agar (V8 agar) with niger seed agar, casein agar and sunflower agar applied to differentiation between C. dubliniensis and C. albicans.

MATERIAL AND METHODS

Strains: a total of 26 Brazilian C. dubliniensis isolates and 93 C. albicans isolates were studied. C. albicans isolates were from the culture collection of the Laboratório de Pesquisas Micológicas, Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria city, RS, Brazil. C. dubliniensis were from different Brazilian cities. All the C. dubliniensis isolates were rigorously identified by phenotypic and genotypic methods8,12,20,21. The strains were stored in frozen stocks at -80 ºC and were routinely propagated on YPD agar plate (10 g yeast extract, 20 g peptone, 20 g glucose, 15 g agar per litre) at 30 ºC13.

Culture media for chlamydospores production: a) Tomato juice agar17 (tomato juice 200 mL; CaCO3 3 g; dextrose 5 g; agar 20 g per liter); b) Niger seed agar20 (50 g Guizotia abyssinica seed pulverized; 1 g glucose; 1 g KH2PO4; 1 g creatinine; 15 g agar per liter); c) Sunflower seed agar3 (extract cooled and filtered from 50 g Helianthus annuus pulverized; 1 g glucose; 1 g KH2PO4; 1 g creatinine and 15 g agar per liter); d) Casein agar14 (10 g of skim milk was dissolved in 90 mL of distilled water, and 3 g of agar was dissolved in 97 mL of distilled water. After autoclaving both solutions separately at 121 ºC for 15 min, they were allowed to cool to 45 ºC to 50 ºC and after this were mixed together. All the media after autoclaving were dispensed in 25 mL amounts into 90 mm-diameter Petri dishes. Tomato juice agar, niger seed agar, sunflower agar and casein agar plates were inoculated with culture growth in a 48 h YPD agar followed by incubation at 30 ºC, except casein agar, which was incubated at 24 ºC. Following the incubation, the plates were directly examined by 10X and 40X objectives lens to observe the chlamydospores. When absent, samples of culture growth were stained with lactophenol cotton blue and were again examined for chlamydospores production by light microscopy.

RESULTS AND DISCUSSION

All the 26 (100%) C. dubliniensis isolates tested produced chlamydospores in Tomato juice agar after 48 h of incubation at 30 ºC. The majority of C. albicans isolates evaluated (92.47%) did not show chlamydospores in the same conditions. However, in the cases that chlamydospores were produced by C. albicans, although indistinguishable from those by C. dubliniensis, were difficult to observe due to their very low number. When we compared the four media for C. dubliniensis identification based on chlamydospore production, we observed that all C. dubliniensis isolates tested (n = 26) produced chlamydospores, yielding 100% of sensibility. On the other hand, when we inoculated all media with C. albicans isolates (n = 93), the production of chlamydospores was very little: tomato juice agar 7/93, niger seed agar 3/93, sunflower agar 3/93 and casein agar 8/93, which have resulted in specificities ranging from 91% to 97%.

C. dubliniensis is usually isolated in mixed cultures with C. albicans and/or other yeasts. The difficulty in detecting mixed cultures in plates with traditional media as Sabouraud dextrose agar has stimulated the development of identification systems and differential media for yeasts1,9,13,17. Specific media is being purposed to discriminate C. dubliniensis from C. albicans such as the niger seed agar (Guizotia abyssinica)20, the sunflower agar (Helianthus annuus)3 and casein agar14 among others2,10,11,19. They are simple and cheap methods for presumptive differentiation of C. dubliniensis from C. albicans, showing satisfactory results. However, it is well established that C. dubliniensis isolates present great phenotypic variability, what hinders the standardization of the identification techniques2,5,7,11,16,21; so the search for new and more refined methods are advantageous and should be stimulated. Here we have studied the tomato juice agar which is a traditional medium employed to observe the formation of ascus to differentiate the sexual reproduction as seen in Saccharomyces cerevisae from the asexual reproduction in yeasts like Candida species1,9,13,17. We noticed that tomato juice agar stimulates the chlamydospore production in C. dubliniensis similarly to what occurs with other media. Chlamydospores, from greek chlamys that means mantle, plus spora that means seed or spore, is a thick-walled thallic conidium that generally functions as a resting spore1. These fungal structures contain very significant reserves which are activated and consumed during germination until the newly formed colony has developed sufficiently for its own trophic extension1,9.

Concluding, we emphasize that tomato juice agar is a well known medium used in Mycology laboratories, simple to prepare, cheap and that now, it can have a double usage indicating the sexual differentiation of yeasts and as a presumptive medium for differentiation between C. albicans and C. dubliniensis.

Received: 15 August 2005

Accepted: 21 February 2006

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  • Correspondence to:
    Sydney Hartz Alves
    Rua Andradas 1985/201
    97010-033 Santa Maria, RS, Brasil
    e-mail:
  • Publication Dates

    • Publication in this collection
      20 July 2006
    • Date of issue
      June 2006

    History

    • Received
      15 Aug 2005
    • Accepted
      21 Feb 2006
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