Biol Res 37: 747-757, 2004

ARTICLES

Genetic polymorphism of clinical and environmental strains of Pichia anomala

EUGENIO REYES^1-2, SALVADOR BARAHONA³, OLGA FISCHMAN¹, MAURICIO NIKLITSCHEK³, MARCELO BAEZA³ and VÍCTOR CIFUENTES³*

¹ Microbiology and Immunology Division -Federal University of São Paulo - Brazil
² Fundación Científica y Tecnológica, Asociación Chilena de Seguridad ACHS
³ Depto. Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile

Dirección para Correspondencia

ABSTRACT

In this work 20 clinical and 3 environmental yeast isolates were characterized by classical morphological and physiological methods, as well as molecular methods based on PCR of the ITS1-5.8S rDNA-ITS2 region. The characteristic morphology and biochemical profiles observed in these samples correspond to those described for the Pichia genera, more specifically to P. anomala. The profiles of susceptibility to five antifungal drugs were determined by two broth dilution methods. The results obtained by both methods were comparable and showed that clinical isolates presented more resistance to azoles, amphotericin B, and 5-fluorocytosine, than environmental ones did. By amplification and sequencing of internal transcribed spacers (ITS1 and ITS2) and the ribosomal 5.8S DNA, the yeast samples were divided into four groups, where the strains within each group had the same sequence. Of the analyzed yeast isolates, 78% were identified as Pichia anomala. Using RAPD analysis with seven different Operon primers, polymorphism was observed within the four groups. Our study highlights the growing importance of P. anomala in fungemic episodes in premature neonates. Furthermore, the methodologies used provide a powerful tool to identify and determine differences in similar strains of this yeast.

Key terms: Pichia anomala, 5.8S rDNA, RAPD, internal transcribed spacer, antifungal drugs.

INTRODUCTION

The ascomycetous yeast Pichia anomala is considered to be a non-pathogenic member of the normal or transient flora of the human alimentary tract and can also be found in plants, soil, fruits, and animals (13, 25, 32, 36, 37, 43). However, it has recently been described in a growing number of clinical cases of infections in patients who present predisposing factors such as lingering hospitalization, immunosuppresion, and wide spectrum antibiotic therapy, as well as increasing catheter use in the prevalence of nosocomial fungal infections. (2, 6, 22, 24, 26- 29, 31, 33, 35, 38, 42, 44, 46, 47). These infections carry a high mortality rate depending on the underlying condition and whether effective antifungal therapy was administered on the fungemic episodes to which they have been associated. At present, little information is available about infections due to P. anomala, and no common treatment with antifungal drugs has been established (1, 6, 34). For an optimal antifungal treatment, a rapid identification of the yeast species causing the acute infection is necessary when the conditions of the human host are still favorable. Identification based on standard medical laboratory techniques (biochemical pattern and morphologic characteristics) that require purification of the target organism is time-consuming and may not be species specific (17, 18). Additionally, strains of the same species can differ in key characteristics (9), and these methods are usually not sensitive enough to identify different strains belonging to the same species.

New molecular methods for a rapid and accurate identification of microorganisms based on chromosomal genes have been developed (11). In this direction, methods based on the polymerase chain reaction (PCR) have been shown to be the most appropriate tools for rapid identification of microorganism, including bacteria, yeast, and microalga, etc. (8, 15, 18, 23). More recently, differences in regions that are less conserved due to less evolutionary constraints, such as the rRNA internal transcribed spacer (ITS1 and ITS2) that separate the conserved 18S and the 28S from the 5.8SrDNA gene, can be used to discriminate species within some genera, and has been used to identify P. anomala from a variety of sources (5,19, 21). In addition, in recent years the utilization of large databases of rDNA and ITS1 and ITS2 sequences are becoming available for all type of fungal species, including P. anomala, which makes the process of identification possible (14, 39).

In the present work, we analyzed and identified 20 clinical yeast samples isolated from blood of premature neonates and 3 environmental isolates. We evaluated the usefulness of combining conventional medical laboratory and molecular biology analysis as a tool for the identification of fungal agents causing infections. Additionally, the susceptibility profile to 5 antifungal drugs commonly used in the treatment of fungal infections was determined. By mean morphological and biochemical characterization, these samples were identified as P. anomala. The 23 yeast strains were characterized by molecular methods using primers ITS1 and ITS4 that target conserved regions of fungal ribosomal RNA. The strains were grouped and identified by comparing the DNA sequences obtained from amplicons. The majority of them were identified as Pichia anomala, corroborating the preliminary identification obtained from laboratory medical methods. The genetic diversity of strains presented in each group was evaluated by RAPD analysis.

MATERIALS AND METHODS

Organisms. A total of 20 clinical and 3 environmental yeast isolates obtained from the yeast collection of the Laboratório de Micologia da Escola Paulista de Medicina (Universidade Federal de Sao Paulo) were analyzed. The clinical samples were originally isolated in 1996 from hemocultures of premature neonates in the Center of Health of Santa Marcelina (Sao Paulo, Brazil). The environmental samples were isolated from orange juice. The yeast strains were grown and maintained on Sabouraud dextrose agar or on YEPD. Routine medical-mycology techniques such as macroscopic characterization, microscopic analysis of the ascospores and biochemical profiles were used to identify primary yeast (17).

Physiological and biochemical characterization. The ability of the yeast isolates to assimilate different carbon and nitrogen sources and ferment carbohydrates was assayed according to standard methods (17).

Antifungal agents. The following five antifungal drugs, supplied by manufacturers as pure standard compounds were used: fluconazole (FLU), amphotericin B (AMB), ketoconazole (KETO), itraconazol (ITRA) and 5-fluorocytosine (5-FC). The antifungal stock solutions were prepared in water (FLU and 5-FC), in polyethylene glycol (KETO), and in dimethylsulphoxide (AMB and ITRA).

Antifungal susceptibility test methods. These determinations were performed by using the broth macrodilution and microdilution reference methods according to the NCCLS document M27-A (30).

i) Broth macrodilution test. The culture medium used was RPMI 1640, with L-glutamine, without sodium bicarbonate, and buffered to pH 7.0 with 0.165 M MOPS. The yeast inoculum suspension was prepared using the spectrophotometric method to obtain a final yeast concentration of 0.5 - 2.5x10³ cells/ml. The assay tubes were incubated at 35 ºC and were inspected 48 h after inoculation. The MIC reading criterion was the lowest concentration that produced at least 80% inhibition of growth, compared to the control tube without an antifungal agent.

ii) Broth microdilution test. The same RPMI-1640 media described above was utilized. The assays were performed in sterile, flat-bottomed 96-well microplates (Nunclon, Delta, Nunc., InterMed, Denmark), containing 100 ml of antifungal solution at twice the final concentration. Each well was inoculated with 100 ml of a yeast suspension adjusted to 0.5 and 2.5 x 10³ cells/ml using the spectrophotometric method. The assay wells were incubated at 35ºC and were inspected 48 h after inoculation.

The final concentration range used of each antifungal agent was as follows: FLU, 0.125 _ 64 mg/ml; 5-FC, 0.125 _ 64 mg/ml; KETO, 0.03 - 16 mg/ml; ITRA, 0.03 _ 16 µg/ml; and AMB, 0.03 - 16 mg/ml. The minimal inhibitory concentration (MIC) for AMB was defined as the lowest concentration of drug that resulted in complete inhibition of visible growth. The MIC for azoles was established as the lowest concentration of antifungal agent which resulted in an 80% reduction of fungal growth compared to the control (drug-free) (3). The break point for MICs of 5-FC, AMB and azoles were those suggested by the NCCLS M27-A (30).

DNA extraction. To isolate genomic DNA, yeast isolates were grown for 48 h at 35 °C in liquid YM medium (yeast extract 0.3%, peptone 0.5%, malt extract 0.3%, glucose 1%). DNA from 50 ml culture was isolated as described (16) with some modifications. Briefly, cells obtained by centrifugation at 10,000 x g for 10 min were resuspended in 5 ml of 0.9 M sorbitol, 0.1 M EDTA and 100 ml of zymoliase 100T (2 mg/ml) was added. The mixture was incubated for 25 min at 37°C, centrifuged at 4,000 x g for 5 min and the pellet was resuspended in 4.5 ml of 50 mM Tris-HCl, 20 mM EDTA. A volume of 500 ml of SDS 10% was added, incubated at 65°C for 15 min and 50 ml of proteinase K was added, followed by incubation at 55°C for 60 min. Two ml of cold 5M potassium acetate was added and incubated on ice for 10 min. One volume of saturated phenol pH 8.0 was added, and both phases were gently mixed. The aqueous phase was recovered and washed twice with 1 volume of phenol:chloroform:isoalmylic alcohol (25:24:21) and once with chloroform: isoamylic alcohol (24:1). The DNA was precipitated with 2 volumes of ethanol at -20°C.

PCR amplification. The primer pairs ITS1 (5´-TCCGTAGGTGAACCTGCG-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) were used to amplify the 5.8S rDNA and the adjacent ITS1 and ITS2 regions (14). PCR amplification was performed in a final volume of 25 ml as follows: 1 ml of yeast DNA (aprox. 10 ng) was added to the PCR master mixture which consisted of 2.5 ml of 10x PCR buffer, 0.5 ml of dNTP's mixture (10 mM of each one), 2 ml of ITS1 and ITS2 primer mix (25 mM of each primer), 1 ml of MgCl₂ (50 mM) and 0.2 ml (1 U) of Taq polymerase. The final volume was adjusted with distilled water. Amplification was performed in a GeneAmp PCR system 2700 thermal cycler (Applied Biosystem) as follows: initial denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s; annealing at 55 °C for 30 s; synthesis at 72°C for 1 min, repeated for 30 cycles; and a final extension step at 72 °C for 10 min (20). Negative control reactions without any template DNA were carried out simultaneously. The amplified products were separated electrophoretically in 1% agarose gels in TAE buffer containing ethidium bromide (0.5 mg/ml) and photographed under transilluminated UV light. The mass of the bands was quantified using the 1D Image Analysis Software version 2.0.1 (Kodak Scientific Imagen System) using as standard the 100 bp DNA Ladder (Fermentas). PCR products were recovered from agarose gels and purified by an alternative to the glassmilk method according to Boyle and Lew (4).

RAPD analysis. RAPD fingerprinting was performed with seven decamer arbitrary primers OP AC-02, OP AC-04, OP AC-06, OP AC-10, OP AC-17, OP AC- 18 and OP AE-20 (Operon Technologies, Inc., Alameda, CA) according to the manufacturer's instructions. For each 25-ml reaction mixture, the reactants were added as follows: 2.5 ml of 10 x PCR Buffer, 2.5 ml of 1 mM dNTPs, 2 ml of 25 mM MgCl₂, 2 ml of primer (10 pmol), 5 ml of 10 pg/ml DNA template, 0.4 ml Taq DNA polymerase (2 U) and distilled water to adjust the final volume. Amplification was performed in a GeneAmp PCR system 270 (Applied Biosystem) programmed as follows: initial denaturation at 3 min 95°C; 1min 94°C for denaturing, 2 min 35°C for annealing; 2 min 72°C for synthesis, repeated for 40 cycles; with a final extension step of 5 min at 72°C. Amplicons were separated on 1% agarose gels in TAE buffer containing ethidium bromide (0.5 mg/ml), photographed under transilluminated UV light and the mass of the bands was quantified as described above. The RAPD bands data was analyzed using the TREECON for windows version 1.3b software (45).

Nucleotide sequence accession numbers. The rDNA nucleotide sequences have been deposited in the National Center for Biotechnology Information (NCBI) GenBank data library under the following accession numbers: clinical isolates: P1, AY349447; P2, AY349450; P3, AY349437; P4, AY349440; P5, AY349435; P6, AY349445; P7, AY349443; P8, AY349455; P9, AY349441; P10, AY349448; P11, AY349454; P12, AY349436; P13, AY349442; P14, AY349438; P15, AY349446; P16, AY349444; P17, AY349456; P18, AY349439; P19, AY349449 and P20, AY349453; environmental isolates: A1, AY349451; A3, AY349452 and A9, AY349457.

RESULTS

Morphological and physiological characterization

Colonies of clinical isolates obtained after incubation at 25°C for 72 h on Sabouraud-dextrose agar presented a bright aspect, plain surface, regular border, cream color, and no pigment. The environmental isolates were granular or serous in texture, with a rough or plain surface, irregular border, white color, and no pigment. In the microscopic characterization, both clinical and environmental isolates presented rudimentary pseudomycelia, with 20% of blastic cells and typical hat-form ascospores. In the sugar assimilation test (Table I), all strains assayed were able to assimilate glucose, galactose, maltose, sucrose, trehalose, mannitol and xylose, and were unable to assimilate melibiose, rhamnose, ducitol and inositol. An assimilation assay for ducitol and inositol has not been reported previously for P. anomala. The assimilation of cellobiose was variable between the strains. All strains assimilated KNO₃, produced esters and formed a film upon growth (data not shown). Additionally, all the strains had the capacity to ferment dextrose, maltose and sucrose sugars with the exception of lactose. Only two clinical strains, P1 and P4, were able to ferment galactose (not shown). The morphological and physiological data obtained suggest that the clinical and environmental yeast isolates belong to the species P. anomala (17)

Susceptibility to antifungal agents

For a better characterization, the susceptibility to antifungal drugs (FLU, KETO, ITRA, AMB and 5-FC) of the 23 yeast isolates was determined by broth macrodilution and microdilution methods. The MIC values obtained for a particular antifungal agent were comparable between the two methods, supporting the NCCLS recommendation that both methods can be used. All environmental isolates were susceptible to the five antifungal drugs assayed. All the clinical isolates were susceptible to AMB, 60% to FLU, 25% to KETO, 30% to ITRA and 25% to 5-FC. Table II summarizes the susceptibilities of 20 clinical and 3 environmental isolates to anphotericin B, 5-fluorcytosine and azoles. AMB showed MIC values homogenously low against clinical and environmental samples, with MIC₅₀ and MIC₉₀ of 0.5 µg/ml for both groups, indicating the high susceptibility to this polyene. In relation to the azoles, the MICs obtained for clinical samples were several times higher when compared to those obtained for the environmental samples, being 8 (FLU), 2 (ITRA) and 4 (KETO) times bigger. 5-FC presented the widest variation in both groups, and clinical isolates presented MIC₉₀ values that were 500 fold higher than environmental ones. When MIC values to azoles are compared, we point out that P. anomala samples presented better resistance to KETO and ITRA than to FLU.

Polimorphism within the ITS element of the rRNA

A region spanning the internal transcribed spacer ITS1 and ITS2, including 5.8S rDNA gene, was amplified from clinical and environmental yeast isolates using primers ITS1 and ITS4 (17). Amplicons obtained were of three different sizes ranging between 400 to 650 bp as determined by agarose gel electrophoresis. Both strands were sequenced and sequences were compared and analyzed. In a preliminary analysis the isolates were divided into four groups as shown in Table III. Group I (582 bp), includes the clinical isolates P8, P11 and P20; Group II (618 bp), was composed by clinical isolates P1, P2, P6, P7, P10, P15, P16, P19, and environmental isolates A1 and A3; Group III (619 bp), includes the clinical isolates P3, P4, P5, P9, P12, P13, P14, and P18; and Group IV (388) is composed of a clinical isolate P17 and environmental isolate A9. DNA sequences for all isolates in each group were identical and were compared to the GenBank database using the BLAST alignment program to identify fungal species with a high level of accuracy (12). The group I isolates were identified as Candida qinlingensis, group II and III as Pichia anomala and group IV as Candida haemulonii, all with 99% identity. Groups II and III presented similar ITS1 (182-183 bp) and ITS2 (191 bp) sequences, suggesting no differences between them, as was the case of yeast isolates belonging to group I that presented ITS1 and ITS2 of 160 and 177 bp length respectively . However, the yeast isolates from group IV had short ITS1 (67 bp) and ITS2 (81 bp) sequences, suggesting that the two isolates of this group do not belong to the Pichia yeast species. The Pileup analysis of the ITS1-5.8S rDNA-ITS2 sequence gave a dendrogram clearly indicating that the first three groups are related, with a high level of similarity between group II and III, while the two yeast isolates belonging to group IV are completely different (Fig. 1) (10). Basically, the same results were obtained when the analysis was performed with only the ITS1 or ITS2 sequence. The length of the 5.8S ribosomal RNA gene was 157 bp in the first three groups and was shorter (153 bp) in group IV isolates, corroborating that the isolates of the last group belong to another species. Furthermore, the comparison of the 5.8S rDNA sequence from all isolates indicated that the sequences of isolates belonging to groups II and III were identical, implicating a high degree of sequence conservation of the 5.8S rRNA gene in the clinical isolates P1 to P7, P9, P10, P12 to P16, P18 and, P19 and in the environmental isolates A1 and A3. Additionally, the clinical isolates P8, P11 and P20 belonging to group I have a high sequence similarity to the yeast isolates from groups II and III and could correspond to Pichia anomala.

Figure 1. Dendrogram of clinical and environmental yeast isolates based on the sequence of ITS elements, including 5.8S rDNA gene. Sequences were analyzed by multiple sequence comparison using a PileUp program of the GCG Wisconsin Package. P1 to P20 correspond to the clinical isolates and A1, A3 and A9 correspond to environmental isolates.

RAPD analysis

A Randomly Amplified Polymorphic DNA (RAPD) analysis was used as a second method of genotyping, to determine differences among the isolates belonging to each of the groups of yeast or to confirm that the isolates within each group were only one strain. A total of 22 different bands were observed using the seven primers in each of the 13 clinical and 2 environmental isolates analyzed. Results obtained using the OPAC-06 primer are shown in Figure 2. The sizes of the RAPD bands observed ranged between 100 to 2,140 bp. A tree constructed from the RAPD data of all seven primers, after UPGMA analysis using the TREECON software version 1.3b, clearly shows that all yeast isolates are polymorphic and are genotypically different (Fig. 3) . In spite of the fact that yeast isolates belonging to each group have identical ITS1, 5.8 rDNA and ITS2 DNA sequences, the RAPD analysis permits the identification of differences between these isolates, indicating that they are genetically different strains. Despite the great diversity observed with the RAPD analysis, all isolates were clustered in two major branches. Branch 1 was composed of eight yeast isolates belonging to groups I (P8), II (P6, P10, P19, P2) and III (P4, P12, P13), and branch 2 was composed by the remaining seven yeast isolates belonging groups II (P16, P7, A3, A1) and III (P5, P18, P14). Within branch 2, a clear separation of the environmental isolates as a sub-branch can be observed.

Figure 2.Pattern of amplified yeast DNA using 10-mer OPAC-06 primer. A total of 13 clinical and 2 environmental isolates were analyzed. Lanes: 1, P7; 2, A1; 3, A3; 4, P14; 5, P5; 6, P19; 7, P16; 8, P4; 9, P18; 10, P6; 11, P10; 12, P12; 13, P3 ; 14, P2; 15, P8 ; and 16, negative control. M, 100 bp ladder (Fermentas).

Figure 3. Dendrogram showing RAPD polymorphism of the 13 clinical and 2 environmental isolates of P. anomala. This tree was constructed with RAPD results obtained with seven 10-mer primer operons (OP AC-02, OP AC-04, OP AC-06, OP AC-10, OP AC-17, OP AC- 18 and OP AE-20) as described in the TREECON software.

DISCUSSION

New fungal infections in humans are being seen with increasing frequency because of the use of multiple antibiotics and other risk factors. The clinical isolates utilized in this study were originally isolated from hemocultures of different premature neonates from the same Health Center during one year. They were identified as P. anomala by morphological and physiological analysis. However, identification of yeast species that have been emergent in recent years has been difficult. For example, the characteristics of P. anomala samples isolated from clinical material and from the atmosphere do not differ from each other and from those observed in other species of yeasts of the Candida genera. The molecular analysis of the ITS1, 5.8 rDNA and ITS2 region, which has been used successfully in the rapid identification of fungi (7, 12, 14, 39), identified only four different groups from the yeast isolates studied. All yeast samples within a group have the same sequence, even though each isolate was obtained from a different patient. Furthermore, some environmental isolates have an identical nucleotide sequence of the rDNA region as the rest of the isolates of the respective group. These results suggest that although the variation of the internal transcribed spacer allows differentiation between species (P. anomala from C. haemulonii), it has not been efficient enough to distinguish between strains from the same species, as has been observed by other authors (40). However, the RAPD analysis proved to be a powerful tool in determining genetic polymorphism between the yeast isolates within each group. In previous work this kind of analysis allowed discrimination at both the species and strain levels, of Hansenula anomala isolated from samples with different geographic and source origin (32). Therefore, the combination of the different methods made the detection of differences between strains of the same species possible. Furthermore, although some of the environmental and clinical yeast isolates were identified as P. anomala, differences in their susceptibilities to antifungal drugs was observed, where the clinical isolates presented more resistance to the antifungal agents assayed. These results strongly suggest that systematic infections with P. anomala must not be empirically treated without further determination of their susceptibility to antifungal drugs commonly used in medical treatments.

ACKNOWLEDGEMENTS

This work was supported by Grant 901-4 from Fundación Científica y Tecnológica, Asociación Chilena de Seguridad (ACHS) and Centro de Biotecnología, Facultad de Ciencias, Universidad de Chile.

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Corresponding Author*: Víctor Cifuentes, Laboratorio de Genética, Depto. de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile. Las Palmeras 3425, Casilla 653, Santiago, Chile, E-mail: vcifuentes@uchile.cl

Received: March 26, 2004. In revised form: September 8, 2004. Accepted: October 6, 2004.