Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2005
Volume 8
Issue 3
Topic:
Biotechnology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Bogus豉wska-W御 E. , Czeszejko K. , Czekaj這-Ko這dziej U. , M璠rala D. 2005. INTRASPECIES DIFFERENTATION OF SACCHAROMYCES CEREVISIAE STRAINS ISOLATED FROM FISH AND THE ODRA ESTUARY, EJPAU 8(3), #16.
Available Online: http://www.ejpau.media.pl/volume8/issue3/art-16.html

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

El瘺ieta Bogus豉wska-W御, Katarzyna Czeszejko, Urszula Czekaj這-Ko這dziej, Dagmara M璠rala
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 Szczeci雟ka, Roztoka Odrza雟ka, 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 Szczeci雟ka within the range of stations 5 and 6.

Fig.1 Map of spancing of sampling station

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 Bogus豉wska-W零 and D鉉rowski [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 manufacturers 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: At/A0 × 100 (At 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 Szczeci雟ka, 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).

Table 1. Characteristics of Examined Stations within the Odra Estuary

Group of stations

No.

Station

Possible source of contamination

The total number of microflora

S.cerevisiae
CFU/mL

Yeasts
CFU/mL

Faecal coliforms
MPN/100mL

Cedynian Land-scape Park

1

Widuchowa

wild birds, wetlands

0.3 x 10

4.0 x 102

0.1

Above Szczecin

2

 

3

Gryfino

 

Higway

Fish farm, post-cooling dumping of waters by

"Dolna Odra" power plant

wetlands, waste-land

0.5 x10

 

1.0 x 10

3.2 x 102

 

3.2 x 102

0.1

 

0.09

Szczecin a municipal area

4

5

6

Duty Bridge

Long Bridge

Maritime office

Fish

sewage effluents

sewage effluents, municipal wastes

sewage effluents

7.5 x 10

3.5 x 103

8.6 x 102

0.2 x 10

9.0 x 102

5.1 x 103

1.8 x 103

1.4 x 102

0.02

0.002*

0.001*

0.6

Roztoka Odrza雟ka

7

 

8

Police

 

Trzebie

Pollution delivered by "Police" chemical plant

municipal wastes, pollution from fishery port

1.25 x 102

 

0.6 x 10

5.2 x 102

 

7.7 x 10

0.02

 

0.01

Pomeranian Bay

9

winoujcie

pollution from the wina river and port

0.0 x 10

2.5 x 10

1

*bc beyond class

Table 2. Characteristic of analyzed strains of Saccharomyces cerevisiae

Signify of strains

No. of strains

Origin

No. of sampling

Sampling station

1/66
1/65
1/67
1/1115
1/1124
3/75
4/68
4/74
4/74a
4/76
4/77
4/78
4/81
4/84
4/90
4/92
4/3/1
4/1116
4/1118
4/1119
4/1123
4/1126
5/68
5/69
5/74a
5/76
5/81
5/84
5/89
5/92
5/93
5/1110
5/1114
5/1115
5/1116
6/71
6/72
6/73
6/85
6/87
6/88
6/89
6/95
6/96
6/1111
6/1112
6/1113
7/83
7/1120
7/1125
7/1127
7/1128
8/12
8/13
8/61
8/62
8/63
8/D5
9/12
9/82
R/1117
R/94
wz/97

65
66
67
1115
1124
75
68
74
74a
77
76
78
81
84
90
92
3/1
1116
1118
1119
1123
1126
68
69
74a
76
81
84
89
92
93
1110
1114
1115
1116
71
72
73
85
87
88
89
95
96
1111
1112
1113
83
1120
1125
1127
1128
12
13
61
62
63
5
12
82
1117
94
97

1
1
1
1
1
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
8
8
8
8
8
8
9
9
4
4

Widuchowa
 
 
 
 
Highway
Duty Bridge
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Long Bridge
 
 
 
 
 
 
 
 
 
 
 
 
Marine Board Base
 
 
 
 
 
 
 
 
 
 
 
Police
 
 
 
 
 
Szczecin Lagoon
 
 
 
 
 
Pomeranian Bay
Fish
 
IHAR W6

Fig.2 Significant differences in faecel coliform /yeasts/ S.cerevisie

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 Szczeci雟ka). 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.

Fig.3. Groups hydrophobicity of S.cerevisiae

Fig.4. Hydrophobicity of S.cerevisiae

Fig.5. Dendrogram demonstrating the genetic relationship isolated of S.cerevisiae

Fig.6. RAPD-PCR fingerprints as representative of 5 main groups (A-E) and unique profiles (U1-U5)

REFERENCES

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  2. Bogus豉wska-W零 E., D鉉rowski W., Frazik M. Hydrofobowo ciany kom鏎kowej dro盥篡 i grzyb闚 dro盥穎podonych w modyfikowanych rodowiskach. 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.

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  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.


El瘺ieta Bogus豉wska-W御
Department of Food Microbiology,
Agricultural University of Szczecin, Poland
Papie瘸 Pawa VI 3, Szczecin, Poland
email: ewas@tz.ar.szcecin.pl

Katarzyna Czeszejko
Department of Food Microbiology,
Agricultural University of Szczecin, Poland
Papie瘸 Pawa VI 3, Szczecin, Poland

Urszula Czekaj這-Ko這dziej
Department of Food Microbiology,
Agricultural University of Szczecin, Poland
Papie瘸 Pawa VI 3, Szczecin, Poland

Dagmara M璠rala
Department of Food Microbiology,
Agricultural University of Szczecin, Poland
Papie瘸 Pawa VI 3, Szczecin, Poland

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