INTRASPECIES DIFFERENTATION OF SACCHAROMYCES CEREVISIAE STRAINS ISOLATED FROM FISH AND THE ODRA ESTUARY

Elżbieta Bogusławska-Wąs, Katarzyna Czeszejko, Urszula Czekajło-Kołodziej, Dagmara Mędrala
Department of Food Microbiology, Agricultural University of Szczecin, Poland

ABSTRACT

Saccharomyces cerevisiae strains isolated from fish and subsurface layers of the Odra Estuary waters were examined. It was evaluated that the increase in faecal contamination of water samples is related to the increase in the total number of yeasts and yeast-like organisms, including S. cerevisiae. RAPD-PCR analysis enabled to distinguish 5 groups of isolates presenting a high level of similarity of their genetic fingerprints. Evaluation of S. cerevisiae cell wall hydrophobic properties, conducted with the MATH method, revealed occurrence of three separate phenotypic groups which expressed the tested property at different level. 87.5% of hydrophobic strains originated from the most polluted area. No correlation between genotypes and hydrophobicity of strains was observed.

Key words: S. cerevisiae, hydrophobicity, randomly amplified polymorphic DNA (RAPD-PCR) technique, water.

INTRODUCTION

The aquatic environment consists of qualitatively and quantitatively varied microflora which creates characteristic structures typical of particular reservoirs. Affected by anthropogenic influences, the aquatic environment becomes a medium for atypical, distinctive groups of microorganisms introduced involuntarily by human industrial and household activities. Such microorganisms may either survive and become an inseparable part of the aquatic biocoenosis or undergo a process of slow extinction. A colonization of atypical aquatic environments forces phenotypic changes in introduced microorganisms by creating new links among them and natural microflora (e.g. biofilms, actually not always based on symbiotic relationships) and/or higher organisms. Microorganisms which replace original microflora may pose serious problems. No competition or antagonistic relationships within the aquatic environment promotes the occurrence of dominant strains which may be a source of waterborne infections for people and animals. Present studies show that each year a significant increase in the number of mycosis cases caused by ‘emerging pathogens’ is observed [6,12]. Surprisingly, considered as non-pathogenic and commonly known and widely used in industry and household Saccharomyces cerevisiae has been included in the group of pathogenic yeasts since the 1990s [5,6,10,11,16]. Entering a particular biocoenosis S. cerevisiae becomes its integral part. The occurrence of this microorganism in new ecological structures may be determined by phenotypic flexibility of strains, e.g. their hydrophobicity, which may reflect their intraspecies differentiation. According to Doyle and Rosenberg [4] expression of cell surface hydrophobicity (CSH) is most frequently a consequence of a cell wall surface structure. Cells of S. cerevisiae are lipophilic due to their proteins [9]. As proteins are responsive to environmental factors their conformational changes cause variations of cell wall properties. CSH of bacteria and yeasts is regarded to be one of the basic mechanisms which enables their colonization thus fundamentally their survival in the environment. It is also a feature known to contribute to pathogenicity of microorganisms [7].

According to that, the aim of the work was both to determine the intraspecies differentiation of S. cerevisiae strains isolated from fish and the Odra estuary at the molecular level and to analyze their cell wall hydrophobicity.

MATERIAL AND METHODS

Source of Microorganisms

Studies covered the Odra estuary which consists of Odra Szczecińska, Roztoka Odrzańska, the Szczecin Lagoon and the Pomeranian Bay (Figure 1). Samples were collected in stations regarding the indexes of hydrochemical pollution [14] and characteristic of potential pollution sources. All samples were collected from the subsurface water layer once a month from April to November, 2001. Fish caught in the Odra Szczecińska within the range of stations 5 and 6.

Isolation and Identification of Yeasts and Yeast-Like Organisms

The evaluation of the total number of yeasts and yeast-like organisms was performed according to Bogusławska-Wšs and Dšbrowski [1]. Isolates from fish were cultured on Sabouraud Dextrose Agar supplemented with chloramphenicol (Oxoid). Species identification was confirmed using biochemical tests API ID 32 C (bioMèrieux).

DNA extraction

DNA was extracted from 24 h cultures in 2 ml of YPD medium at 30°C using QIAamp DNA Mini kits (Qiagen) according to the manufacturer’s instructions. Efficiency of extraction was evaluated during electrophoresis in a 1% agarose gel (Prona Agarose Plus).

Oligonucleotides and RAPD-PCR Assay

8 primers/set of primers were applied displaying different levels of differentiation potential. Among them the set of RP1-4 (5' TAG GAT CGA A 3') and SOY (5' AGG TCA CTG A 3') was chosen as the most promising [8]. Optimization of the assay was performed according to an orthogonal array designed by Taguchi and Wu [13] and modified by Cobb and Clarkson [3]. The PCR was performed in a volume of 20 µL containing 500 mM KCl, 100 mM Tris-HCl pH 8.3 (at 25°C), 3.5 mM of MgCl₂, 0.625 mM of dNTP, 12.5 pmol/mL of each primer and 1U of Taq DNA polymerase (Eppendorf) and 20 ng/µL of template DNA in Mastercycler Gradient (Eppendorf). The thermal profile consisted of one initial cycle: denaturation at 95°C, annealing at 36°C, elongation at 72°C performed for 5 min each and followed by 30 cycles of a 94°C denaturation for 1 min, a 72°C annealing for 1 min and a 72°C elongation for 2 min. At the end of amplification the mixture was subjected to the final extension at 72°C for 10 min. Products were analyzed by electrophoresis in 2% agarose gel (Prona Agarose Plus) stained with ethidium bromide (0.5 µL/mL) in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3) and examined under UV light (GelDoc, BioRad). Quantity One (BioRad) and BioGene (Vilber Lourmat) software were used to estimate the similarity (Dice 2%) within a group of isolates.

Hydrophobicity Tests

Yeast strains were cultured as described earlier [2]. The MATH (Microbial Adhesion To Hydrocarbons) method was applied to evaluate CSH of strains [15]. Changes in absorbance of strain suspensions in relation to a non-polar solvent – hexadecane – were measured by using a “Carlzeiss” spectrophotometer at 600 nm. According to a formula: A_t/A₀ × 100 (A_t – preliminary extinction of suspension, A₀ – extinction of suspension after defined time of vortexing measured in relation to a blank sample – phosphorous buffer) obtained results enabled to create a curve which was a determinant of microorganism affinity to hexadecane.

Fecal Contamination of Water

Index of fecal contamination of water was calculated according to the PN-77 C-04615 standard and also with regard to the ISO 7251:1993 standard.

Statistical Analysis

Analysis of dispersion describing correlation between separate groups of microorganisms was performed at a confidence limit 95%. Analysis of concentrations of S. cerevisiae strains depending on their hydrophobicity was carried out by a single-link method counted by Euclidean distance (Statistica PL). Adjusting a function in relation to CSH intensity was performed by the smallest square method whereas significance of variations between hierarchic groups was confirmed by Scheffe test at the confidence limit p<0.05.

RESULTS

Analysis of distribution of tested microflora revealed that the most polluted area was an urban part of the Odra Szczecińska, including stations nos. 5 and 6 (Table 1). Statistical comparison showed that the presence of S. cerevisiae strains as well as the total number of yeasts and yeast-like fungi in tested samples was directly proportional to the index of E. coli contamination (Figure 2). Correlation between the total number of yeasts and yeast-like organisms and the number of S. cerevisiae isolates was confirmed (Figure 2). Only two strains of S. cerevisiae were isolated from fish (strain nos. 5/1117 and 5/94) (Table 2).

Based on statistical analysis two main hierarchical levels describing the degree of S. cerevisiae hydrophobicity were found. Two groups: hydrophilic - B (65% of strains) and hydrophobic - A (34% of strains) were determined. The hydrophobic group was divided into two separate subgroups presenting CSH described as highly hydrophobic and intermediately hydrophobic - A1 and A2, respectively (Figure 3). Based on the statistical adjusting of the function significantly differed sections were assigned characteristic of CSH of examined strains (Figure 4). No statistical correlation between the occurrence of strains and their hydrophobicity was found. However, 87.7% of hydrophobic strains was isolated from the most polluted area. It comprised 39% of the total number of S. cerevisiae strains isolated from the most polluted stations where samples were collected (the Odra Szczecińska). No correlation between genotypes and hydrophobicity of strains was observed.

RAPD-PCR analysis of isolates revealed occurrence of 5 groups of isolates (A-E) presenting a high level of similarity of their genetic fingerprints (70-100%). Five unique strains (U1-U5) were also found (Figures 5 and 6). The persistent occurrence of clonally related strains of the same RAPD type along the sampled area of the river gave a clear evidence of transmission of potentially epidemic and endemic S. cerevisiae strains highly adapted to particular conditions. RAPD-PCR technique revealed a high differentiation potential and proved to be notably useful in the environmental studies of typing and monitoring S. cerevisiae distribution.

REFERENCES

1. Bogusławska-Wšs E., Dšbrowski W, 2001. The seasonal variability of yeasts and yeast-like organisms in water and bottom sediment of the Szczecin Lagoon. Int J Hyg Environ Health 203: 451-458.

2. Bogusławska-Wšs E., Dšbrowski W., Frazik M. Hydrofobowoœć œciany komórkowej drożdży i grzybów drożdżopodonych w modyfikowanych œrodowiskach. EJPAU (in press).

3. Cobb B.D., Clarkson J.M., 1994. A simple procedure for optimizing the polymerase chain reaction (PCR) using modified Taguchi methods. Nucleic Acids Res 22: 3801-3805.

4. Doyle R.J., Rosenberg M., 1993. Microbial cell surface hydrophobicity. American Society for Microbiology, Washington D.C.

5. Garcia-Martos P., Dominguez I., Marin P., Garcia-Agudo R., Aoufi S., Mira J., 2001. Antifungal susceptibility of emerging yeast pathogens. Enferm Infecc Microbiol Clin 19: 249-256 (abstract).

6. Hazen K.C., 1995. New and emerging yeast pathogens. Clin Microbiol Rev 8: 462-478.

7. Hazen K.C., Wu J.G., Masuoka J., 2001. Comparison of the hydrophobic properties of Candida albicans and Candida dubliniensis. IAI 69:779-786.

8. Lehmann P.F., Lin D., Lasker B.A., 1992. Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymophic DNA. J Clin Microbiol 30:3249-3254.

9. Lesser C.F., Miller S.I., 2001. Expression of microbial virulence proteins in Sacchraomyces cerevisiae models mammalian infection. EMBO J 20: 1840-1849.

10. Ponton J., Ruchel R., Clemons K.V., Coleman D.C., Grillot R., Guarro J., Aldebert D., Ambroise-Thomas P., Cano J., Carrillo-Munoz A.J., Gene J., Pinel C., Stevens D.A., Sullivan D.J., 2000. Emerging pathogens. Med Mycol 38:225-236.

11. Sobel J.D., Vazquez J., Lynch M., Meriwether C., Zervos M.J., 1993. Vaginitis due Sacchraomyces cerevisiae: epidemiology, clinical aspects, and therapy. Clin Infect Dis 16: 93-99 (abstract).

12. Swoboda-Kopeć E., Rokosz A., Sawicka-Grzelak A., Wróblewska M., Krawczyk E., Stelmach E., Łuczak M., 2001. Etiological agents of fungemias in hospitalized patiens. Med Doœw Mikrobiol 53: 291-295.

13. Taguchi G., Wu Y., 1980. Introduction to off-line quality control. Japan Quality Control Organisation. Nagoya Japan.

14. WIOS, 1999. The report on the condition of the environment in Western Pomeranian region in 1999. The Nature Preservation Inspection. The Regional Nature Preservation Inspectorate in Szczecin. The Library of the Environmental Monitoring. Szczecin.

15. Van der Mei H.C., de Vries J., Busscher H.J., 1993. Hydrophobic and electrostatic cell surface propierties of thermophilic dairy streptococci. Appl Environ Microbiol 59: 4305-4312.

16. Xu J., Boyd C.M., Livingston E., Meyer W., Madden J.F., Mitchell T.G., 1999. Species and genotypic diversities and similarities of pathogenic yeasts colonizing women. J Clin Microbiol 37: 3835-3843.

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