Susceptibility to heat and antifungal agents of Cryptococcus neoformans var. neoformans (serotype D) isolated from Eucalyptus spp in Rio Grande do Sul, Brazil

Susceptibilidade de Cryptococcus neoformans var. neoformans (sorotipo D) isolados de Eucalyptus spp., no Rio Grande do Sul (Brasil), frente ao calor e a agentes antifúngicos

MEDICAL MICROBIOLOGY

Susceptibility to heat and antifungal agents of Cryptococcus neoformans var. neoformans (serotype D) isolated from Eucalyptus spp in Rio Grande do Sul, Brazil

Susceptibilidade de Cryptococcus neoformans var. neoformans (sorotipo D) isolados de Eucalyptus spp., no Rio Grande do Sul (Brasil), frente ao calor e a agentes antifúngicos

Jorge A. Horta^I; Josiane Faganello^I; Lívia K. Rosa e Silva^I; Loiva T. Oliveira^II; Jânio M. Santurio^II; Marilene H. Vainstein^I; Sydney Hartz AlvesII,^*

^ICentro de Biotecnologia, Programa de Pós-graduação em Biologia Celular e Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil

^IIDepartamento de Microbiologia e Parasitologia, Laboratório de Pesquisas Micológicas, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil

ABSTRACT

In this work we studied the susceptibility to heat and antifungal agents of the first strains of environmental Cryptococcus neoformans var neoformans (serotype D) isolated in the state of Rio Grande do Sul, Brazil. In order to achieve a rigorous analysis, we employed the methodology recommended by NCCLS, Yeast Nitrogen Base (YNB) proposed by Ghannoum et al (YNB-1), Antibiotic medium 3 (AM3) indicated by others, YNB adjusted to the NCCLS methodology (YNB-2) and Etest. Our results indicate that all strains were susceptible to amphotericin B (0.0625-0.5 µg/mL), fluconazole (0.125-8.0 µg/mL), itraconazole (0.031-0.25 µg/mL) and flucytosine (0.125-4.0 µg/mL). The C. neoformans serotype D strains were more susceptible to heat (47ºC/30 min) than C. neoformans serotype A.

Key words:Cryptococcus neoformans, heat susceptibility, antifungal agents

RESUMO

Este estudo foi realizado com os primeiros isolados ambientais de C. neoformans sorotipo D, obtidos no Rio Grande do Sul. Objetivando-se avaliar a susceptibilidade a agentes antifúngicos de forma mais rigorosa, utilizou-se a técnica de referência proposta pelo NCCLS, Caldo Yeast Nitrogen Base (YNB) proposto por Ghannoum et al., Antibiotic medium 3, caldo YNB adequado à metodologia do NCCLS e o E-test. Os resultados indicaram que todos os isolados foram sensíveis à anfotericina B (0,0625-0,5 µg/mL), fluconazol (0,125-4,0 µg/mL), itraconazol (0,031-0,25 µg/ml) e fluorocitosina (0,125-4,0 µg/mL) através das técnicas empregadas. Nos testes de termotolerância (47ºC/30 min), observou-se que as culturas de C. neoformans sorotipo D são mais sensíveis do que as de C. neoformans sorotipo A.

Palavras-chave:Cryptococcus neoformans, susceptibilidade, antifúngicos

INTRODUCTION

Cryptococcus neoformans, an encapsulated yeast, is the etiological agent of human cryptococcosis. In Brazil, 4.5% of all opportunistic infections in AIDS patients have been reported as being caused by C. neoformans (23). Based on physiological and serological differences, C. neoformans had been divided in two varieties: C. neoformans var. neoformans (serotypes A, D, AD) and C. neoformans var. gattii (serotypes B and C). On the basis of genetic differences it was proposed that C. neoformans var. neoformans be further subdivided in two varieties: C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D) (12).

The prevalence of the varieties and serotypes from either clinical or environmental sites differs in accordance with geographical localization. Environmental isolates of C. neoformans var. grubii from pigeons droppings indicate that serotype A is more common than serotype D in all nations, except Italy, Denmark and Switzerland (8) and have been recovered from approximately 99% of all patients in most countries (6,8,12). The serotype C is rarer than the other serotypes and has never been isolated from the environment. The majority of the few clinical isolates of serotype C have been found in southern California (8). The serotype B was initially isolated from Eucalyptus camaldulensis and E. tereticornis in Australia and more recently it has been isolated from other trees and immunocompromised (non-AIDS) patients in tropical and subtropical areas (8). In Rio Grande do Sul, C. neoformans var. grubii (serotype A) has been recovered from AIDS patients and pigeons excreta (1,6,14). The prevalence of C. neoformans var. neoformans (serotype D) among clinical isolates has ranged from 0 to 100% depending on the region of the world. In Brazil, the prevalence of serotype D is very low (29), and in the state of Rio Grande do Sul there were no records of clinical C. neoformans var. neoformans (serotype D) isolates.

Recently Ribeiro (28) isolated nine C. neoformans var. neoformans (serotype D) strains from 99 samples of Eucalyptus spp. As pointed out by Martinez et al. (21), the thermal sensibility of C. neoformans var. neoformans (serotype D) is a consistent explanation for the geographic differences between serotype A and D, and for the dermatotropism and rhinotropism observed. The purpose of this investigation was to assess the susceptibility of the first environmental C. neoformans var. neoformans (serotype D) isolates in Rio Grande do Sul, to heat and to a panel of antifungal agents commonly used in the treatment of infections. Because results of susceptibility tests based on M27-A2 methodology have been questioned due to factors such as suboptimal growth in RPMI 1640 medium and narrow amphotericin B MIC ranges (15,20), we decided to compare amphotericin B MICs obtained with RPMI 1640 medium to assays performed using Yeast Nitrogen Base proposed by Ghannoum et al. (YNB-1)(13), Antibiotic medium 3 (AM3) and Yeast Nitrogen Base supplemented medium adjusted to the M27-A2 methodology, which we named YNB-2 and the Etest method.

MATERIALS AND METHODS

Isolates

Porto Alegre, the capital city of the South Brazilian state Rio Grande do Sul is situated at 30º S latitude and 51º W longitude. The city is 10 m above the sea level with an average temperature of 22ºC and an annual rainfall of 1118 mm. Ninety-nine samples were collected from different Eucalyptus species (Table 1). Twenty other environmental strains of C. neoformans serotype A, isolated from pigeon excreta from Porto Alegre, Santa Cruz do Sul and Santa Maria (14), were also included in the analysis of heat susceptibility. All isolates were identified as C. neoformans by positive Niger seed agar response, as well as urease test, ability to grow at 35ºC and a negative nitrogen test. The profiles of carbon compound assimilation were also determined (4). Canavanine-glycine-bromothymol blue agar medium, assimilation of D-proline and D-tryptophan were used for differentiation of the two varieties (9) and serotyping was performed by a slide agglutination test (Crypto Check; Iatron Co; Japan).

Susceptibility to heat (21)

A 1-ml aliquot of cells suspended at a density of 10⁴ cells per ml in distilled water was incubated at 47ºC in water bath during 30 min. After incubation 20 µL of the cells suspension was plated onto Sabouraud dextrose agar and incubated for 48h at 30ºC. The survival percentage was determined by comparison to non-heat-treated control samples plated onto Sabouraud dextrose agar.

Antifungal susceptibility tests

The antifungal agents used were amphotericin B (Sigma), fluconazole (Pfizer), itraconazole (Jansen) and flucytosine (Sigma).

Test media were: RPMI 1640 (American Biorganics Inc.), indicated by the National Committee for Laboratory Standards (NCCLS) as reference, and Yeast Nitrogen base (Difco) were prepared according to manufacturer instructions. After reconstitution, both media were supplemented with glucose to obtain a final concentration of 0.5% in YNB (13) and 2% in RPMI 1640 (24). Both RPMI 1640 medium and YNB were buffered to pH 7.0 with 3-(N-morpholino) propanesulphonic acid (MOPS; Sigma, St. Louis, Mo, USA) to a final concentration of 165mM (NCCLS). Antibiotic medium 3 (AM3) (BBL) was supplemented with glucose to a final concentration of 2%; the buffering capacity was increased by adding 1g of dipotassium monophosphate per liter and 1g of monopotassium monophosphate per liter and pH was adjusted to 7.0 with NaOH (20). All three media were filter sterilized by using 0.22-µm membrane (Millipore).

The MICs of the tested agents were determined for each isolate in accordance with National Committee Laboratory Standards (NCCLS) macrodilution guidelines (24). The tubes were incubated at 35ºC and were read after 48h of incubation. The MIC of amphotericin B in RPMI 1640, YNB and AM3 was defined as the lowest concentration of drug that resulted in complete inhibition of visible growth. The tests were performed using two techniques with YNB: YNB-1 as indicated by Ghannoum et al. (13) and YNB-2 which use the same broth, but using the M27-A2 method (NCCLS). The MIC of azoles and flucytosine in RPMI 1640 were determined according to M27-A method (24). The data were reported as MIC ranges and MICs at which 50% and 90% of these isolates were inhibited. Quality control testing was performed in accordance with NCCLS document M27-A2. Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 (24) were used as quality control for the susceptibility tests.

Etest

To prepare the agar plates, the double-strength, filter-sterilized RPMI 1640 with 2% glucose was buffered with potassium phosphate at pH 7.0 and combined with an equal volume of heat-sterilized double-strength agar to yield the correct final concentration of medium in a 1.5% agar gel. The inoculum suspensions of C. neoformans strains matching the turbidity of McFarland #1 standard were swabbed onto the surface of the agar plate and allowed to dry for 15 min before the addition of the Etest strip (22). One Etest antimicrobial gradient strip was placed in each Petri dish. The plates were incubated for 48 and 72h, and the MIC was the point at which the zone of complete inhibition intersected the strip. Etest antimicrobial gradient (AB Biodisk, Solna, Sweden) strips containing amphotericin B, fluconazole, itraconazole and flucytosine were employed (32).

RESULTS

All isolates of C. neoformans var. neoformans (serotype D) grew well in RPMI 1640 broth, YNB-1, YNB-2, Antibiotic medium 3 and RPMI-1640 agar, allowing MICs to be determined after 72h incubation.

Table 2 summarizes the in vitro susceptibilities of the nine cultures tested by NCCLS method. The results are reported as MIC ranges, MIC₅₀s (50% of strains were inhibited) and MIC₉₀s (90% of strains were inhibited). A broad range of MICs was observed with fluconazole and flucytosine; more narrow MIC ranges were showed with amphotericin B and itraconazole. Table 3 summarizes the in vitro susceptibilities of the isolates to amphotericin B, as determined by NCCLS recommended medium, and other media suggested in the literature. All media employed showed similar MIC ranges, but YNB-1 showed slightly higher results. The MIC₅₀ and MIC₉₀ were the same for RPMI-1640, AM3 and YNB-2, but again higher for YNB-1. Based on the M27-A2 technique breakpoints, all the isolates were considered to be sensitive to the antifungal agents tested.

The MICs obtained by Etest are shown on Table 4. Itraconazole MICs are narrower in range, but amphotericin B, fluconazole and flucytosine had broad range of MICs. Based on MIC_50% or MIC_90%, all the isolates were considered to be sensitive to the antifungal agents tested.

Among the 9 serotype D strains tested, 5 (55.5%) did not survive heat treatment and 4 (44.5%) showed percent survival varying between 22 and 76% when compared to the number of colonies in an equivalent suspension of cells not exposed to heat. When the 20 serotype A strains were assayed, only 3 (15%) did not grow on Sabouraud Dextrose agar after the thermal treatment; the range of percent survival was from 16 to 86%. The average percent survival of serotype D was 23.2%, while for serotype A it was 48%.

DISCUSSION

An understanding of the epidemiology of cryptococcosis may provide a rational framework for the design of prevention guidelines and more effective therapies. We believe that attempts to correlate specific environmental exposures to cryptococcal strains of known susceptibility profile may help to address some questions (14).

Based on these epidemiological statements, it is important to emphasize that cryptococcosis therapy may become even more difficult due the emergence of antifungal resistance. Amphotericin B (17,19), fluconazole (3,25,26,27), and flucytosine resistance have been well documented (30). Cross-resistance to both azole-types and amphotericin B has also been described (16).

In addition, recent articles reporting primary (intrinsic) resistance of C. neoformans var. neoformans (serotype D) to flucytosine (18) and, the isolation of fluconazole-resistant C. neoformans from an immunocompetent patient, without prior exposure to azoles (25), emphasizes the importance of carrying out susceptibility tests before beginning of therapy. This study reports the susceptibility testing of the first C. neoformans serotype D isolated from Eucalyptus spp. trees in Rio Grande do Sul state, employing additional methods for more accurate delineation of susceptibility profiles to antifungal agents.

The results of susceptibility tests to antifungal agents reported here are similar to those reported by others authors (7,15). However, some aspects deserve attention. In 2001, we have compared the susceptibility of clinical and environmental C. neoformans isolated in southern Brazil and observed that clinical isolates were less susceptible to fluconazole than environmental isolates, all of them of serotype A (1). Here, environmental strains showed a very similar pattern for amphotericin B and azoles, being all strains sensitive. One of the reasons to the absence of azole resistance might be the source of strains, which may have not had previous contact with azoles. The contrary has been reported in Italy, where clinical serotype D is prevalent. Tortorano et al. (31) showed that serotype A clinical strains were less susceptible to fluconazole than serotype D clinical isolates. This issue may deserve more attention.

The reference M27-A2 method based on RPMI 1640 medium has been used to test C. neoformans isolates (1,2,10,20). In spite of this indication, some previous reports suggested that RPMI 1640 medium did not support suitable growth of C. neoformans. In the present study, we have found that this medium, supplemented with 2% glucose, provided adequate growth of all strains tested; glucose supplementation is an alternative mentionated in the M27-A2 method (24). Due to reported concerns regarding amphotericin B resistance, in this study we have tested Antibiotic medium 3 (AM3), as indicated by Lozano-Chiu et al. (20), Yeast Nitrogen Base, as recommended by Ghannoum et al. (13), and also the same medium with changes in inoculum size and endpoint determination (YNB-2). The results presented have shown that the pattern of susceptibility to amphotericin B obtained with YNB-2, RPMI-1640 medium and AM3 was closely similar, with overlap of MIC ranges. YNB-1 resulted in a broader MIC range, though amphotericin B resistant strains were not detected. These results are similar to those already obtained from clinical strains of C. neoformans serotype A using the same media (2).

The MICs to flucytosine were low and so, based on established breakpoints, all strains were considered to be sensitive to this drug. This finding must be interpreted with caution, because approximately 2% of C. neoformans isolates are resistant to flucytosine prior to treatment (30). So, we must consider that the number of isolates studied was scarce, and DNA studies have shown that Brazilian C. neoformans isolates appeared to be less heterogenous than those isolated from other regions (11).

The Etest method performed on glucose-supplemented RPMI 1640 agar is an excellent method of discrimination between susceptible and resistant strains of C. neoformans (20,22,32). The susceptibility pattern obtained by Etest demonstrated a narrow range MICs to amphotericin B, and MIC₅₀ and MIC₉₀ one log dilution higher than the NCCLS method. For all antifungal agents studied, we have observed no significant changes on MICs by this method. This is in accordance with previous studies, that found complete or a high level of agreement between Etest and NCCLS method (5,22,32). The Etest has been considered an excellent method to distinguish amphotericin-B-resistant yeasts; it is reproducible, much simpler to set up than broth dilution methods and less labour-intensive. Thus, it has been recommended for routine use with amphotericin B and flucytosine (32).

Recently, Martinez et al. (21) analysed the heat susceptibility of 19 strains from each serotype group and observed a wide variation; C. neoformans serotype D strains being more susceptible. Our finds are in accordance with this study and corroborate what Dromer et al. (8) pointed out in that the differences in the prevalence of serotype A and D of C. neoformans may reflect climatic tolerances. The isolation of C. neoformans var. neoformans (serotype D) in Rio Grande do Sul (28), the southernmost and coolest state of Brazil, reflects the characteristics referred above, and might explain the rarity of this serotype in other areas of Brazil with sub-tropical and tropical climates. However, few studies were conducted and data are still scarce in this area (29).

Submitted: August 25, 2003; Returned to authors for corrections: July 07, 2004; Approved: March 28, 2005

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Publication Dates

• Publication in this collection
  12 Sept 2005
• Date of issue
  Mar 2005

History

• Received
  25 Aug 2003
• Accepted
  28 Mar 2005