Cell surface hydrophobicity and its relation to adhesion of yeasts ...

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Cell surface hydrophobicity and its relation to adhesion of yeasts isolated from fish gut. R. V6zquez-Juk-ez”*b,*, T. Andlid”, L. Gustafssona. aDepartme& of ...

Colloids and Surfuces B: Biomterfaces, 2 (1994) 1999208 0927-7765194/$07.00 0 1994 ~ Elsevier Science B.V. All rights reserved.


Cell surface hydrophobicity and its relation to adhesion of yeasts isolated from fish gut R. V6zquez-Juk-ez”*b,*,

T. Andlid”, L. Gustafssona

aDepartme& of General and Marine Microbiology University of GSteborg, Carl Skotsbergs Gata 22, S-41 3 19 GSteborg, Sweden bCentro de hvestigaciones Biologicas de Baja California Sur, Mhco, P.O. Box 128, La Paz B.C.S.. M&co


24 March

1993; accepted

10 July 1993)

Abstract Five different yeast strains isolated from fish. Succharomyces cereowze HFl and F2. Sc182 (unidentified stram), Rhodotorula rubrn and Rhodotorula g[utims, were used in this study. The cell surface hydrophobtcity (CSH) was dependent on growth m all cases Exponential-phase cells were always hydrophobic while stationary-phase cells became hydrophilic. In contrast, two laboratory strains, Saccharomyces cerecwae Y41 and Debaryomyces hansenii 526 behaved in the opposite manner as prevrously reported for S cereoisme and Candida albicans This fact, together with mtcroscoptc observations, prompts the suggestion that the cessatton of populatton growth (budding) leads to hydrophilic cell differentiation. Irrespective of the degree of hydrophobicity of the surface examined, exponential-phase cells dtd adhere to a greater extent than stationary-phase cells. These results suggest that hydrophobicity plays an important role m viva. Since hydrophobic interactions have been suggested to be the most important forces mediatmg attachment in the aquatic environment, we do believe that these forces mediate the mittal events of fish colonizatton by yeast strains Key wlords’Adhesion;

Cell surface hydrophobtctty;

Fish gut yeasts

Introduction Cell surface hydrophobicity

(CSH) in the aquatic

environment is of major importance for microbial cells. Although there are several attraction forces mediating the attachment of cells to inert and biotic surfaces, hydrophobic interactions are probably the most important [ 11. The ecological meaning of the CSH of aquatic microorganisms can be explained as the advantage of attachment and colonization of surfaces by cells that simultaneously require aqueous media for growth and development [ 21. The CSH of Candida albicans has been the focus of interest for medical microbiologists since it was correlated with virulence and also with participa*Corresponding


SSDI 0927-7765(93)01067-2

tion in the adhesion of yeast cells to biomaterials [336]. The basis for increased virulence is related to the adhesion of yeasts to the tissue or host cells [7]. Nevertheless, the strongest mechanism for adhesion seems to be specific adhesin-receptor binding, with CSH being perhaps the dominating that contributes to adhesion. initial force Moreover, CSH has been proposed as favouring the accuracy of adhesin-receptor bonds and thereby enhancing the number of successful contacts between two surfaces [S]. In another animal model, the CSH of the fish pathogen Renibacterium salmoninarum is virulence related when tested against rainbow trout Salmo gairdneri [9]. The adhesion to plastic surfaces has been described as occurring in three phases [6]: (i) adsorption to the liquid-plastic interface; (ii) initial or reversible adhesion to the plastic surface;

and (iii) irreversible

or permanent


to the

plastic surface. CSH could be considered to be the force that accounts for the reversible phase, which greatly


properties, prevailing

the final adhesion.


are highly


The adhesion



on the

and the phe-

Deburyornyces FFIJWS



controls. Detroit,


[ 131 and


The growth


38531) were used as was YNB (Difco,

MI) plus 0.5% glucose,

methionine and histidine


0.5% (NH,)$O,,

(20 mg l- ’ )_ trypophan

(20 mg l- ‘)

( 10 mg 1-l). YNB with an increased

notypic state of the cells [6]. Characteristically. CSH is not a rigidly preserved property of the cell envelope but is, rather, dynamically controlled to

C/N ratio (glucose (20 g 1-l) and ( NH,)$O, (0.0618 g 1-l )) was used to induce lipid accumulation during the cultivation of Rh. glutinis. The

suit environmental changes and physiological conditions [ l,lO,ll]. There is a considerable amount of information relating to the CSH of bacterial cells and its physiological and ecological meaning. Except for the role of CSH in the pathogenesis of C. albicans,

CSH was also measured after growth in YPD (1% yeast extract. 2% peptone and 2% glucose) as well as after growth in crude mucus. Precultures were prepared by overnight growth in the experimental media except for the experiments in mucus, where precultures were prepared in YPD.

there is a noticeable lack of information about yeast cells. The role of the cell surface properties of aquatic yeasts, particularly in population dynamics, has so far been ignored. Some yeast strains have recently been demonstrated to colonize in large numbers the intestine of rainbow trout without any visible negative effects on the fish [12]. With the goal of understanding the mechanisms of yeast colonization, and in the long run developing an effective probiotic strain, the growth in and the adhesion to mucus, as colonizing factors, were also examined. Both of these were found to be positive [ 121. In order to obtain more information on the mechanisms of yeast adhesion. the CSH of yeasts from fish under different conditions were examined in the present

work. The dynamics

of CSH are eval-

uated and discussed in comparison with the behaviour of other groups of microorganisms. Likewise. the influence of CSH on the adhesion to different surfaces is examined.


of crude FOCUS

Fresh rainbow trout were kept on ice during transport from the fish farm. The fish were aseptically opened and the intestine was cut free. The section of the intestine from the pylorus downwards. including the rectum. was separated and opened with sterile scissors. The lumen was intestinal content with sterile rinsed from HEPES-HANKS buffer (pH 7.2). The mucus was carefully scraped from the intestinal wall with a with rubber scraper, collected in a tube HEPES-HANKS, and vortexed to dissolve the mucus material. Finally, the mucus was centrifuged twice at 23000g for 1.5 min to remove undissolved material such as epithelia and microbial cells, and was stored at -70°C. Each batch of mucus was collected from 7-8 fish to avoid individual differences. Determination

of cell swfuce


Materials and methods Preparation

of cell suspensions

and growth


Five yeast strains, Succhuromyces cerevisiae HFl and F2, Sc182 (unidentified strain), Rhodotorulu rubru and Rhodotorulu glutinis, isolated from fish intestine, were used in this study [ 121:

The hydrophobic microsphere assay (HMA) described by Hazen and Hazen [ 141 was used with some modifications. The glassware was soaked overnight in 1% HCl, rinsed with Milli-Q water and heated to 17O’C for 3 h. Culture samples were collected and washed twice with phosphatebuffered saline (PBS) and adjusted to 1 x 10’

R. Vrizque:-Judrer

et al IColloids

Surfaces B. Biomeyfaces

,’ ( 1994 ) 199--30X

cells ml-’ of PBS. From a stock colloidal suspension (IO% solids) of polystyrene latex, 6 ul of bluedyed microspheres, 0.8 urn in diameter, (Serva Fine Biochemicals, Westbury, NY) were mixed with 2 ml of PBS (pH 7.2) at 4°C. Equal volumes (50-100 ~1) of the yeast cell suspension and the microsphere suspension were mixed in glass tubes, equilibrated for 1 min at room temperature and vortexed for 30 s. Microsphere attachment was assayed immediately by optical microscopy. The percentage of cells with three or more attached microspheres was recorded as the hydrophobic proportion of the population. At least 100 cells were counted per assay. When clusters of cells and microspheres were observed, together with a few single cells without microspheres, more than 95% hydrophobicity was assumed. In elucidating the dynamics of CSH, yeasts were cultured in a fermenter (BiofIo, New Brunswick Inc.) with a working volume of 0.5 1. The temperature was 28”C, the stirring rate was 300 rev mini and the aeration rate was 2 I air min-‘. The pH was controlled at 4.5 by the automatic addition of 0.1 M NaOH. Growth was followed by using a flow-through microcalorimeter (Bioactivity monitor LKB 2277, Thermometric, Jarfalla, Sweden). The culture was pumped with a peristaltic pump (Microperpex pump LKB 2132 LKB-Pharmacia, Uppsala) at a rate of 80 ml h-’ to a T-connection where it was mixed with a Aow of water-saturated air (30°C) at a rate of 40 ml h- ‘. The mixed culture-air flow was pumped through the microcalorimeter, and then returned to the fermenter (for further details see Ref. 15). Labelling of cells and adhesion to surfaces

Three surfaces showing different characteristics were chosen: plastic scintillation vials (Mini Poly-Q vial, Beckman), circular polystyrene wells (Multidish 24 wells. Nunc, Denmark) and glass vials (6 ml glass vials, Packard, The Netherlands). The hydrophobicity of the surfaces was measured with an NRL contact-angle goniometer (RamCHart Inc., NJ). A water droplet was applied to the


surfaces and the contact angle 4 was measured, resulting in values of 79”. 48” and 13? for plastic vials, polystyrene wells and glass vials respectively. Since the surface free energy is proportional to cos y. according to Young’s equation [ 161 it is possible to use q directly as a parameter indicating the surface free energy. The lower the surface free energy, the higher the 4 value and hydrophobicity. Cells were grown in YNB plus 0.5% glucose and 0.5% (NH,),SO,, supplemented with 3H-methionine to a concentration of 5 uCi ml-‘. After 24 and 72 h, cells were harvested. washed twice with HEPES-HANKS buffer (pH 7.2), and finally adjusted to 1 x 10” cells ml-’ in the same buffer. The cell suspension was used immediately in the assay by adding 250 ul to glass scintillation vials, wells of a microtitre polystyrene tissue culture plate and plastic vials. The lower areas of these different test systems were not significantly different. Vials and tissue trays were incubated at room temperature for 2 h; thereafter the culture was aspirated and surfaces were washed twice with HEPES-HANKS buffer. Attached cells were collected by adding 2 x 200 ~1 of 5% sodium dodecyl sulfate (SDS) solution. The activity of the solutions was measured with a liquid scintillation counter (Beckman LS 3801) and the adhesion was expressed in per cent activity of the attached cells in compa~son with the total activity of the cell suspension used in the assay. Lyticase treatment

The cells were grown overnight; thereafter the cells were hydrophobic (100% CSH), and were then harvested and washed twice with PBS. One millilitre of a cell suspension adjusted to lo8 cells ml-’ was centrifuged and resuspended in the same volume of a solution containing 100 units lyticase (p-1.3 glucanase, Sigma) per millilitre ethylenediaminetetra acetic acid, disodium salt 0.1 mM and 1 mM phenylmethylsulfonyl fluoride. The assay mixture was incubated at 37°C for different times. After incubation, the cells were washed and used for CSH measurement employing the HMA.


microcalorimeter. growth

Cell surfuce hydrophobicity Strains isolated Sc182, Rh. rubra

of yeast strains

control strains were tested for CSH after 24 and 72 h of growth in YPD and YNB. Strains from less hydrophobic

after 72 h

of growth (i.e. stationary phase) in both media. although yeasts grown in YNB were seen to be more hydrophobic (Table 1). However. both D. hansenii J26 and S. cereoisiae Y41 increased in CSH during the stationary phase. During exponential growth, these strains were clearly more hydrophobic in YPD than in YNB. Dynamics of CSH during grobr’th of strainsfio?n$sh The expression of CSH during different phases of growth was evaluated in Rh. glutinis, Rh. rubra and S. cerevisiae F2, growing in a fermenter with a working volume of 0.5 1 of YNB. For Rh. glutinis, the medium was modified to induce lipid accumulation as described in the section Materials and Methods. The production of heat was measured on line by connecting the fermenter to a flow

Table 1 Cell surface hydrophobuzlty (i SD) of yeasts Isolated from fish and collectlon strams m different media and growth phases Stram

Rh. gltctvi? Rh. rubrub S. crrecisu HFl b Sc182b D hunsenii J26d S. cererisiae Y41d

Cell surface


YPD 24 h

12 h

84 * 3.39 83i2.86 68 + 1.69 _c

32 * 4.32 95

100 >95 100 100 0




“Percentage of population latex beads (see Materials bStram from fish. “Not detected. dCollectlon strain.


us to follow changes

bmdmg three or more hydrophobic and Methods sectlon).


(i.e. caused

by depletion of some substrate or metabolic All three strains expressed a high CSH

from fish S. cerevisiae HFl, and Rh. glutinzs and the two

fish were consistently


and localize metabolic

shifts). during

exponential growth. but a decreasing CSH after the growth had ceased (Figs. l-3). In the case of the oleaginous yeast Rh. glutinis, CSH successively decreased


the lipid



which occurred after nitrogen exaustion (Fig. I). showing that the hydrophobicity of Rh. glutinis was not correlated to lipid accumulation [ 121. Comparative studies were performed during growth in mucus (Fig. 4). A small-scale device (15 ml of mucus diluted with HEPES-HANKS buffer) was implemented and connected to the microcalorimeter. Even though hydrophobicity was not as high as when the cells grew in YNB, the pattern was similar. In other words, the cells showed the highest level of CSH during the growth phase while CSH declined

after growth


Adhesion sf Rh. rubra to polystyrene Hydrophobic

cells of Rh. rubra were tested for

adhesion to polystyrene. Adhesion was shown to be saturable (Fig. 5) and time dependent, since the number of adhering cells increased for approximately 45 min, after which a constant level was reached (Fig. 6). The rate of adhesion was higher at 8

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