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Applied and Environmental Microbiology, May 2002, p. 2610-2613, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2610-2613.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Thermodynamic Analysis of Growth Temperature Dependence in the Adhesion of Candida parapsilosis to Polystyrene

Amparo M. Gallardo-Moreno,¹ M. Luisa González-Martín,^1* Ciro Pérez-Giraldo,² Eugenio Garduño,² José M. Bruque,¹ and Antonio C. Gómez-García²

Department of Physics,¹ Department of Microbiology, Extremadura University, 06071 Badajoz, Spain²

Received 10 September 2001/ Accepted 27 February 2002

Top Abstract Introduction References

 
The purpose of this work was to study the adhesion to polystyrene of two Candida parapsilosis strains, grown at 22 and 37°C, in terms of hydrophobicity, surface charge, and interaction free energy. Growth temperature changed the surface properties of microorganisms, yielding a good correlation between thermodynamic predictions and adhesion behavior.

Top Abstract Introduction References

 
Candida parapsilosis has emerged as an important nosocomial pathogen (16, 20, 24) with clinical implications, all of which usually occur in association with invasive procedures or prosthetic devices (27). Colonization by candidas and subsequent infection is achieved by a combination of specific (ligand-receptor interactions) and nonspecific mechanisms (1), allowing the yeast to attach to a wide range of tissue types and nonbiological surfaces. This process depends on the cell surface properties of both the host and the fungus (4).

The main nonspecific forces acting during the initial stages of the adhesion process have a thermodynamic nature, and they are mainly related to the hydrophobic effect (11). The contribution of the electrostatic forces to adherence has been questioned (12).

Quantification of surface hydrophobicity and surface charge can be made by different methods (3, 21, 26), but the obtained results are greatly influenced by the environmental conditions in which cells are grown or suspended in experimental procedures (7, 8, 9, 10, 19).

The aim of this work is to study the hydrophobicity and surface charge of C. parapsilosis in relation to the process of adhesion to polystyrene, in order to determine whether the thermodynamic theory predicts the adherence. The effect of growth temperature on the above properties has been also analyzed.

C. parapsilosis strain 294 and C. parapsilosis strain 289 were isolated from blood of patients of Infanta Cristina Hospital (Badajoz, Spain). Yeasts were stored at -80°C and cultured in Sabouraud broth at 22 and 37°C for 48 h. After culture, the yeasts were harvested by centrifugation, 5 min at 1,000 x g (Sorvall TC6; Dupont, Newtown, Conn.), and washed three times in deionized water, potassium phosphate buffer (KPi) (0.01 mol liter^-1) or phosphate-buffered saline (PBS) (0.1 mol liter^-1) depending on the liquid employed in the experimental hydrophobicity assay. Finally, the cells were resuspended in the appropriate fluids.

Water, formamide, and diiodomethane contact angles (_W, _F, and _D, respectively) on lawns of partially dried yeasts were determined using the sessile drop technique (3) (Table 1). Briefly, microorganisms suspended in demineralized water were layered onto 3-µm-pore-size filters (Millipore; Molsheim, France) using a negative pressure. The filters were left to air dry for 30 min and introduced into an environmental chamber, which was allowed to saturate with vapor from the liquid employed. The images were taken as has been described previously (15).

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TABLE 1. Contact angles for C. parapsilosis strain 294, C. parapsilosis strain 289, and polystyrene, and zeta potentials of the fungusa

Once the contact angles were measured, the Lifshitz-van der Waals (^LW) and acid-base (^AB) surface tension components were obtained from the application of the Young-Dupré equation (equation 1) to each probe liquid (25) (Table 2):

(1)

where is the surface tension of the probe liquid, the subindex Y indicates yeast, and ^- and ⁺ denote the electron-donor and electron-acceptor parameters of the AB component, .

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TABLE 2. Lifshitz-van der Waals and acid-base surface free energy parameters and electron donor and electron acceptor components of C. parapsilosis strains 294 and 289 and polystyrene, and probe liquidsa

The total interaction free energy between microorganisms and polystyrene (P) through water (W) is calculated by the sum of the LW and AB interactions (equation 2) (25):

(2)

where

(3)

and

(4)

The zeta potential () of yeasts, suspended in KPi or PBS, was determined through electrophoresis with a Laser Doppler Velocimeter Coulter DELSA 440 (Langley Ford Instruments, Amherst, Massachusetts), applying the Helmholtz-Smoluchowski equation (equation 5) (21):

(5)

and being the viscosity and the dielectric permitivity, respectively, of the suspending liquid and µ_e the electrophoretic mobility.

The adhesion to polystyrene was examined using the technique described by Klotz et al. (13). Yeasts were suspended in KPi or PBS. Volumes of 200 µl were then added to the wells of polystyrene microtiter plates (Greiner; Frickenhausen, Germany), and the average initial optical density (OD₀) was measured with a microtiter plate spectrophotometer (Labsystem Multiscan Plus, Helsinki, Finland). The cells were allowed to adhere for 5, 10, 15, and 20 min in order to study the initial adhesion process. Adherent microorganisms were detected by removing the nonadherent cells by aspiration and washing carefully with PBS or KPi three times. The wells were then filled with 200 µl of PBS or KPi, and their optical density (OD_t) was measured. Finally, the relation [(OD_t OD₀^-1)100] was calculated to determine the percentage of adhering cells.

A paired Wilcoxon test was applied in order to detect if the adhesion differences were statistically significant (P < 0.05).

All the results were obtained in triplicate with independent yeast cultures.

According to water contact angle data (Table 1), as an indicator of hydrophobicity, strain 294 appeared more hydrophobic than strain 289 at both culture temperatures. Although C. parapsilosis often behaves as the nearest neighbor to Candida albicans and Candida tropicalis (17), the hydrophobicity of both strains studied varied with temperature differently from many strains of C. albicans and C. tropicalis, which appear hydrophobic when grown at temperatures below 26°C and mostly hydrophilic at temperatures around or above 37°C (2, 9, 10). Our strain 294 was more hydrophobic when the temperature increased, while strain 289 presented practically no changes in hydrophobicity despite the change in the growth temperature (Table 1). The hydrophobicity is positively related to the number of cells adhering to polystyrene for both strains (Fig. 1 and 2). The more hydrophobic strain adheres more avidly to polystyrene than the hydrophilic one, regardless of the suspending liquid used (see differences between Fig. 1a and b and Fig. 2a and b; P = 0.0078). In strain 294 the water contact angle changes due to increased growth temperature (Table 1) are also well correlated with the increased percentage of cells adhering to polystyrene for both suspending fluids (P = 0.001) (Fig. 1a and 2a). These results concur with the opinions of some authors, who consider the cellular surface hydrophobicity the main molecular force for microorganism adhesion to substrata (6, 18, 22).

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FIG. 1. Average percentage of yeasts adhering to polystyrene during the first minutes of adhesion. Strain 294 (Fig. 1a) and strain 289 (Fig. 1b) were grown at 22 and at 37°C and finally suspended in KPi.

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FIG. 2. Average percentage of yeasts adhering to polystyrene during the first minutes of adhesion. Strain 294 (Fig. 2a) and strain 289 (Fig. 2b) were grown at 22 and at 37°C and finally suspended in PBS.

In addition to cell surface hydrophobicity, the interaction free energy (Table 3) gives a thermodynamic interpretation of the adhesion process. In order to clarify this dependence, the initial adhesion rate, calculated as the linear regression slope of the data for the first 20 min (Fig. 1 and 2), was plotted against the total interaction free energy values (Fig. 3). The lowest interaction free energy values are well correlated with the highest initial adherence to polystyrene, as thermodynamically predicted. Although the increase in yeast adhesion to polystyrene wells is linked to the decrease of absolute value in the interaction free energies, the fact that positive values of were related to an important adhesion to polystyrene indicates that besides the thermodynamic force, other factors must act on adhesion between microorganisms and substrata (5, 13).

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TABLE 3. Lifshitz-van der Waals (GLW), acid-base (GAB), and total interaction free energies (GTOTAL) between C. parapsilosis (strains 294 and 289) and polystyrene (P) through water (W) when yeasts (Y) are incubated at 22 and 37°Ca

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FIG. 3. Relation between the initial adhesion rate and the total interaction free energies for C. parapsilosis. The filled and opened symbols denote cells suspended in KPi and PBS, respectively.

Regarding the electrical contribution to the adhesion process, the increase in the ionic strength for each sample gave a decrease in the absolute zeta potentials (Table 1), implying a rise in the average percentage of cells adhering to the negatively charged polystyrene (23) for strain 294 (P = 0.0234) (Fig. 3). In strain 289, the adhesion was so small that the detection of changes associated with KPi or PBS was probably influenced by large experimental errors (P = 0.9453). The positive relation between suspending medium ionic strength and microorganism adhesion to negatively charged substrata has been previously described (14). What appeared clear was that electrical interaction did not seem to govern yeast adhesion (12), since similar zeta potentials did not correlate with analogous adhesion values (Table 1 and Fig. 1 and 2).

 
We are grateful to the Junta de Extremadura-Consejería de Educación, Ciencia y Tecnología, and the Fondo Social Europeo for the Ph.D. grant awarded to A.M.G.-M., for financial support, and for the IPR99C016 project. We also thank the DGES for the PB97-0378 project and Fondo de Inverstigación Sanitaria, Spain (FIS 00/0293).

 
* Corresponding author. Mailing address: Department of Physics, Extremadura University, 06071 Badajoz, Spain. Phone: 34 924 289532. Fax: 34 924 289651. E-mail: mlglez{at}unex.es.

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Applied and Environmental Microbiology, May 2002, p. 2610-2613, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2610-2613.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gallardo-Moreno, A. M. Articles by Gómez-García, A. C. Search for Related Content PubMed PubMed Citation Articles by Gallardo-Moreno, A. M. Articles by Gómez-García, A. C. Agricola Articles by Gallardo-Moreno, A. M. Articles by Gómez-García, A. C.