Langmuir 2002, 18, 3639-3644
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Comparative Study of the Hydrophobicity of Candida parapsilosis 294 through Macroscopic and Microscopic Analysis A. M. Gallardo-Moreno,† A. Me´ndez-Vilas,† M. L. Gonza´lez-Martı´n,*,† M. J. Nuevo,† J. M. Bruque,† E. Gardun˜o,‡ and C. Pe´rez-Giraldo‡ Department of Physics and Department of Microbiology, Extremadura University, 06071Badajoz, Spain Received November 15, 2001. In Final Form: February 4, 2002 Several studies have been developed on the hydrophobicity of different strains of Candida albicans, but little is known about the hydrophobic characteristics of Candida parapsilosis, despite the fact that this species is becoming an important pathogen. This work shows a study of the influence of the incubation temperature on the hydrophobicity of C. parapsilosis strain 294, through macroscopic techniques based on contact angles measured on deposited lawns of yeasts and on cell adhesion to solvents hexadecane and chloroform (microbial adhesion to solvents (MATS) test) and through a microscopic technique by using atomic force microscopy (AFM). All of the techniques employed have shown that the yeast surface becomes more hydrophobic when grown at 37 °C than at 22 °C. However, some discrepancies have been found between the behavior of yeasts against hexadecane or chloroform in MATS and the interfacial free energy predictions between the yeasts and each of those liquids calculated from contact angle measurements. On the other hand, AFM techniques have indicated a different temperature influence on the surface properties of the two kinds of patches that lateral force microscopy reveals. Also, all techniques have displayed a similar sensibility to detect the changes induced in the surface characteristics by the incubation temperature, but in this case, AFM shows a different behavior for the different surface patches, too.
Introduction Candida species are the most common fungal opportunistic pathogens encountered in clinical medicine.1,2 Although Candida albicans has been studied extensively because of its large implications in medical infections,3,4 it must be taken into account that other Candida species,5,6 such as Candida parapsilosis,7,8 are being considered as important nosocomial pathogens. C. parapsilosis has clinical implications including fungemia,9 endocarditis,10 endophthalmitis,11 septic arthritis and peritonitis;12 all of them usually occur in association with invasive procedures or prosthetic devices.12 It is widely accepted that the colonization of biomaterials by microorganisms has an initial step in which the physicochemical surface properties, such as hydrophobic* To whom correspondence should be addressed. Tel: +34 924 289532. Fax: +34 924 289651. E-mail:
[email protected]. † Department of Physics. ‡ Department of Microbiology. (1) Odds, F. C. Candida and candidosis. A review and bibliography; Bailliere Tindall: London, 1988; p 10. (2) Klotz, S. A.; Drutz, D. J.; Zajic, J. E. Infect. Immun. 1985, 50, 97. (3) Masuoka, J.; Hazen, K. C. Microbiology 1997, 143, 3015. (4) Millsap, K. W.; Bos, R.; Busscher, H. J.; van der Mei, H. C. J. Colloid Interface Sci. 1999, 212, 495. (5) Rodrigues, A. G.; Mardh, P. A.; Pina-Vaz, C.; Martinez-de-Oliveira, J.; Fonseca, A. F. Infect. Dis. Obstet. Gynecol. 1999, 7, 222. (6) Samaranayake, Y. H.; Wu, P. C.; Samaranayake, L. P.; So, M. APMIS 1995, 103, 707. (7) Turkal, N. W.; Baumgardner, D. J. J. Am. Board. Pract. 1995, 8, 484. (8) Millsap, K. W.; Bos, R.; Busscher, H. J.; van der Mei, H. C. J. Colloid Interface Sci. 1999, 212, 495. (9) Levin, A. S.; Costa, S. F.; Mussi, N. S.; Basso, M.; Sinto, S. I.; Machado, C.; Geiger, D. C.; Villares, M. C.; Schreiber, A. Z.; Barone, A. A.; Branchini, M. L. Diagn. Microbiol. Infect. Dis. 1998, 30, 243. (10) Darwazah, A.; Berg, G.; Faris, B. J. Infect. 1999, 38, 130. (11) Fekrat, S.; Haller, J. A.; Green, W. R.; Gottsch, J. D. Cornea 1995, 14, 212. (12) Weems, J. J., Jr. Clin. Infect. Dis. 1992, 14, 756.
ity, of both interacting phases are the main factors that mediate the subsequent attachment2,3,13,14 usually enhanced by a second step based on specific adhesin-ligand interactions.13 However, many authors have tested that different growth conditions, such as temperature, provoke important changes on the physicochemical surface properties, specially on the cell surface hydrophobicity. A large number of papers present that the hydrophobicity of different strains of C. albicans decreases with increasing the culture temperature15,16 changing, in some cases, from hydrophobic to hydrophilic when the temperature get higher.8 Hydrophobicity has its origin in the relatively ordered structure of water molecules, induced by intermolecular hydrogen bonding.17 Yeast hydrophobicity can be identified by the contact angle that a water droplet makes on a lawn of partially dehydrated yeasts18 or by the number of adhered cells to a hydrocarbon phase.19 But it can be also understood as an interplay of long-range Lifshitz-van der Waals (LW) forces and short-range acid-base (AB) interactions.20-22 In this context and with the assumption (13) An, Y. H.; Friedman, R. J. Handbook of bacterial adhesion. Principles, methods and applications; Humana Press: Totowa, NJ, 2000. (14) Fukazawa, Y.; Kagaya, K. J. Med. Vet. Mycol. 1997, 35, 87. (15) Hazen, B. W.; Hazen, K. C. FEMS Microbiol. Lett. 1989, 48, 167. (16) Blanco, M. T.; Blanco, J.; Sa´nchez-Benito, R.; Pe´rez-Giraldo, C.; Mora´n, F. J.; Hurtado, C.; Go´mez-Garcı´a, A. C. Microbios 1997, 89, 23. (17) Israelachvili, J. Intermolecular and Surface Forces; Academic Press: London, 1992. (18) Busscher, H. J.; Weerkamp, A. H.; van der Mei, H. C.; van Pelt, A. W. J.; de Jong, H. P.; Arends, J. Appl. Environ. Microbiol. 1984, 48, 980. (19) Rosenberg, M.; Gutnick, D.; Rosenberg. E. FEMS Microbiol. Lett. 1980, 9, 29. (20) van Oss, C. J. Colloids Surf., B 1995, 5, 91. (21) Bos, R.; Busscher, H. J. Colloids Surf., B 1999, 14, 169. (22) Azeredo, J.; Visser, J.; Oliveira, R. Colloids Surf., B 1999, 14, 141.
10.1021/la011675y CCC: $22.00 © 2002 American Chemical Society Published on Web 04/02/2002
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that electrical interactions can be neglected,23 the Lifshitz-van der Waals and acid-base surface tension components of both the microorganisms and substrata will allow the interaction free energy of the process to be obtained.24 A novel method to analyze the interaction between microorganisms and substrata is based on the use of an atomic force microscope (AFM).25-27 The AFM is a recently developed instrument which permits mapping the surface topography from the micro- to nanometer scales, in a wide range of conductive and nonconductive samples. Topographic measurement is the most immediate application of the AFM, but it is also able to measure the change in force acting on the tip while it is coming into or retracting from contact with the surface, referred to as the forcedistance curve, which provides information on the adhesion forces, related to the interaction free energy.26-28 Due to the extraordinary high lateral resolution of this technique, the AFM is able to analyze the above changes on different zones of the cell wall. This means that this assay can be employed to make distinctions of thermodynamic magnitudes not only between different strains or species but even between different parts of a cell surface, making possible, at the same time, the detection of changes associated with different culture environments. It seems clear that when different methods are employed in order to study the microorganism surface, based on the atomic description or based on the macroscopic analysis of the cell wall (such as contact angle), the data obtained can give a better understanding of what is happening on the cell surface in relation to the adhesion mechanisms. In this context, this work is aimed to the study of the hydrophobicity of C. parapsilosis strain 294 incubated at two different temperatures, 22 and 37 °C, through a thermodynamical analysis based on contact angles and adhesion to hexadecane and chloroform and through atomic force microscopy. Experimental Section Yeast Strains and Growth Conditions. C. parapsilosis (strain 294) was isolated in hemoculture from patients of Infanta Cristina Hospital (Badajoz, Spain). Yeasts were stored at -80 °C and cultured in Sabouraud (Oxoid, Basingstoke, U.K.) at two different temperatures, 22 and 37 °C, during 48 h. After culture, yeasts were harvested by centrifugation, 5 min × 1000g (Sorvall TC6, Dupont, Newtown, USA) and washed three times in deionized water, for experiments with the AFM and contact angles, and in phosphate-buffered saline (PBS, 0.1 mol L-1) for experiments of adhesion to hexadecane and chloroform, to simulate the physiological conditions when yeasts adhere to a hydrophobic substratum inside the human body. Contact Angle Measurements. Deionized water (Milli-Q Plus), formamide (puriss > 99.0%, Fluka, Switzerland), and diiodomethane (purum > 98%, Fluka) contact angles (ϑW, ϑF, and ϑD, respectively) on lawns of partially dried yeasts were determined using the sessile drop technique.18 Briefly, microorganisms suspended in deionized water were layered onto 3 µm pore size filters (Millipore, Molsheim, France) using a negative pressure. Filters were left to air-dry for 30 min; this time was checked previously as enough to ensure the so-called “plateau contact angles”. After that, filters were placed in an environ(23) Klotz, S. A. FEMS Microbiol. Lett. 1994, 120, 257. (24) van Oss, C. J. Interfacial Forces in Aqueous Media; Marcel Dekker: New York, 1994. (25) Marti, O. In Handbook of Modern Tribology; Bhushan, B., Ed.; CRC Press: Boca Raton, FL, 2000. (26) Weisenhorn, A. L.; Maivald, P.; Butt, H.-J.; Hansma, P. K. Phys. Rev. B 1992, 45, 11226. (27) Nie, H.-Y.; Walzak, M. J.; Berno, B.; McIntyre, N. S. Langmuir 1999, 15, 6484. (28) Nie, H.-Y.; Walzak, M. J.; Berno, B.; McIntyre, N. S. Appl. Surf. Sci. 1999, 144-145, 627.
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Figure 1. Optical density, log[(AtA0-1) × 100], as a function of the vortexing time of C. parapsilosis strain 294. Yeasts were cultured at two different temperatures and suspended in PBS. They were allowed to adhere to hexadecane when grown at 22 °C (b) or 37 °C (9) and to chloroform when grown at 22 °C (O) or 37 °C (0 ). mental chamber G211 (KRU ¨ SS, Hamburg) saturated with vapor of the liquid employed for each of the contact angle measurements, and a drop of the liquid was put on the filter; images of the drop were taken with a video camera and analyzed as has been described previously.29 Contact angle measurements were carried out in triplicate with separately grown yeasts at room temperature. MATS. The microbial adhesion to solvents test (MATS)30 is a modification of the microbial adhesion to hydrocarbons test (MATH)31 to consider kinetic effects32 and to reveal, even calculate, the acid-base contribution in the adhesion process.30 In this context, two different solvents with the same LW component and different AB component are needed. In this work, n-hexadecane (Sigma Chemical, St. Louis, MO; γLW ) 27.7, γ) 0.0, γ+ ) 0.0 mJ m-2) and chloroform (Mallinckrodt Inc., Kentucky; γLW ) 27.7, γ- ) 0, γ+ ) 3.8 mJ m-2) have been employed as hydrophobic substrata to this purpose.22 Briefly, yeasts were suspended to an optical density, A0 (at 600 nm), from 0.4 to 0.6 in PBS. Next, 150 µL of hexadecane or chloroform was added to 3 mL of yeast suspension, and the two-phase system was vortexed for 10 s and then allowed to settle for 10 min. Finally, the optical density At of the suspension was measured. The latter procedure was repeated six times and log[(AtA0-1) × 100] was plotted against the vortexing time for each system in Figure 1. Experiments were repeated three times with separately cultured yeasts at room temperature. AFM Technique. The yeasts, after being suspended in water and diluted, were fixed on previously acid-cleaned glass slides. This sample was fixed to a standard stainless steel planchet with a double-sided sticky tape and mounted in the scanner for subsequent AFM analysis. An Autoprobe CP atomic force microscope (Park Scientific Instrument, Geneva, Switzerland), equipped with a scanner of maximum ranges of 100 µm in the x and y directions and 7 µm in the z direction, was used. The images were acquired by using silicon nitride cantilevers with a nominal force constant of 0.4 N m-1 and a typical probe curvature radius of 10 nm, as supplied by the manufacturer. The scanner speed ranged between 1 and 5 µm s-1, and the images were acquired at 512 × 512 pixels and processed by a secondorder flattening routine. The maximum applied force was ca. 17 nN. Also, force versus distance curves have been collected over the whole surface. Due to the yeast’s shape, the most peripherical regions of the cell were avoided, because measurements in these (29) Moreno del Pozo, J. Ph.D. Thesis, Extremadura University, Badajoz, Spain, 1994. (30) Bellon-Fontaine, M. N.; Rault, J.; van Oss, C. J. Colloids Surf., B 1996, 7, 47. (31) Rosenberg, M.; Gutnick, D.; Rosenberg, E. FEMS Microbiol. Lett. 1980, 9, 29. (32) Lichtenberg, D.; Rosenberg, M.; Scharfman, N.; Ofek, I. J. Microbiol. Methods 1985, 4, 141.
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Table 1. Water (TW), Formamide (TF), and Diiodomethane (TD) Contact Angles for C. parapsilosis Strain 294 and Lifshitz-van Der Waals (γLW) and Acid-Base (γAB) Surface Free Energy Components and Electron-Donor (γ-) and Electron-Acceptor (γ+) Parameters of This Straina
C. parapsilosis strain 294
Tculture
θW (mJ m-2)
θF (mJ m-2)
θD (mJ m-2)
γLW (mJ m-2)
γ(mJ m-2)
γ+ (mJ m-2)
γAB (mJ m-2)
22 °C 37 °C
38 50
42 54
62 59
19.6 21.5
46.0 40.5
4.0 1.4
27.3 15.1
a The average standard deviation over three separate cultures of microorganisms came to (2° for contact angles, (1.1 mJ m-2 for γLW, (2.5 mJ m-2 for γ-, and (0.4 mJ m-2 for γ+.
Table 2. Lifshitz-van Der Waals (∆GLW), Acid-Base (∆GAB), and Total Interaction Free Energies (∆GTOTAL) between C. parapsilosis Strain 294 and Hexadecane (H) and Chloroform (C) through Water (W) When Yeasts (Y) Are Incubated at 22 °C and 37 °Ca Tculture C. parapsilosis strain 294
22 °C 37 °C
LW ∆GYWH (mJ m-2)
AB ∆GYWH (mJ m-2)
TOTAL ∆GYWH (mJ m-2)
LW ∆GYWC (mJ m-2)
AB ∆GYWC (mJ m-2)
TOTAL ∆G YWC (mJ m-2)
0.3 0.0
-13.2 -25.7
-12.9 -25.7
0.3 0.0
-19.9 -30.9
-19.6 -30.9
a The average standard deviation was (0.4 mJ m-2 for ∆GLW and (3.6 mJ m-2 for ∆GAB being mindful of the error propagation of the surface tensions.
regions could be affected by some artifacts by the change in the contact area between tip and sample.
interactions:24 AB ∆GTOTAL ) ∆GLW adh adh + ∆Gadh
Results The average water, formamide, and diiodomethane contact angles on strain 294 are summarized in Table 1. Water and formamide contact angles are higher on yeasts cultured at 37 °C than on those grown at 22 °C, while the diiodomethane contact angle can be considered as not dependent on the sample. Once the contact angles are known, it is possible to evaluate the surface tension of yeasts. The surface tension of a solid or a liquid, γ, can be split into two components, γ ) γLW + γAB, one of them coming from Lifshitz-van der Waals forces (γLW) and the other from acid-base interactions (γAB), and the acid-base component can be written as the geometrical mean24 of its parameters electron acceptor, γ+, and electron donor, γ-, γAB ) 2(γ +γ -)1/2. From the measured contact angles of three different liquids, two polar and one apolar, the surface tension of yeast and its components and parameters can be determined from the three-equation system coming from the application of the Young-Dupre´ equation to each probe liquid:24 LW + - + γL(cos ϑL + 1) ) 2xγLW Y γL + 2xγY γL + 2xγY γL (1)
where the subindex Y indicates yeast and L denotes each one of the liquids used: water (γLW ) 21.8, γ- ) 25.5, γ+ ) 25.5 mJ m-2), formamide (γLW ) 38.8, γ- ) 39.6, γ+ ) 2.3 mJ m-2), and diiodomethane (γLW ) 50.8, γ- ) 0.0, γ+ ) 0.72 mJ m-2).33 The LW and AB components of the surface tension and the electron-donor and electron-acceptor parameters calculated from eq 1 are listed in Table 1. There are not significant changes in γLW components between both temperatures, but the electron-donor and electron-acceptor parameters are higher at the lowest incubation temperature which makes γAB the greatest in this case. Using the yeast’s surface tension and components, the total interaction free energy between yeasts immersed in the suspended liquid (assumed as water for this purpose, because it does not affect the comparative results of this work) and substrata (hexadecane or chloroform) has been calculated (Table 2) by the sum of the LW and AB (33) Janczuk, B.; Wo´jcik, W.; Zdziennicka, A. J. Colloid Interface Sci. 1993, 157, 384.
(2)
where LW LW 2 LW LW 2 ∆GLW adh ) (xγY - xγS ) - (xγY - xγL ) -
LW 2 (xγLW S - xγL ) (3)
and + + ∆GAB adh ) 2[xγL (xγY + xγS + xγL ) + xγL (xγY +
xγ+S + xγ+L ) - xγ-Y γ+S - xγ+Y γ-S ]
(4)
In all cases, the LW component is lower, in absolute value, than the AB component, which means that the theoretical adherence will be governed by short-range forces. At each temperature, the AB contribution to the surface free energy is higher for chloroform than for hexadecane. The negative values of the ∆GTOTAL indicate, from a thermodynamic point of view, a favorable adhesion for both solvents and growth temperatures. Figure 1 shows the dependence of log[(AtA0-1) × 100] against the vortexing time when yeasts, suspended in PBS, were allowed to adhere to hexadecane and chloroform. After 40 s of vortexing, curves of adhesion to chloroform indicate an increase of log[(AtA0-1) × 100] while those to hexadecane do not seem to get stationary values. These behaviors, obtained after a long exposure of cells to such aggressive liquids, could indicate that results are not very confident in this zone. Then, we evaluate the initial removal rate R0 (min-1) as a measurement of the adhesion of the cells by a linear least-squares fitting to the initial section of these lines. The values found for this coefficient are 3.3 and 4.5 min-1 for the adhesion to chloroform for yeast incubated at 22 and 37 °C, respectively. In the case of the adhesion to hexadecane, the initial slopes were 1.2 and 2.0 min-1 for yeast incubated at 22 and 37 °C, respectively. For a given solvent, the initial rate of adhesion is higher for cells cultured at 37 °C than at 22 °C, and for a given growth temperature, it is higher to chloroform than to hexadecane, probably due to the presence of the electron-acceptor parameter in the former solvent. There are various modes of operation in the AFM technique depending on the way the tip interacts with the
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Figure 3. Force vs distance curves in different regions of the yeast cell surface (37 °C): (a) the spot zone and (b) the rest of surface.
Figure 2. AFM images (5 × 5 µm2) of the yeast cultured at 37 °C: (a) forward LFM image (from left to right); (b) backward LFM image (from right to left).
sample. In contact mode, the tip is brought into continuous contact with the sample and raster-scanned over the surface. A feedback loop maintains a constant deflection between the cantilever and the sample, or in other words, the force between tip and sample remains constant. However, during scanning the cantilever not only bends along the normal to the surface but cantilever torsion (lateral) deformation is observed. Lateral force microscopy (LFM) measures this deformation during scanning in contact mode.34 With this technique, it is possible to distinguish the areas of the sample with different friction, so that LFM is sensitive to the composition of the surface.35,36 Generally, LFM and topography images are collected simultaneously. Parts a and b of Figure 2 show the forward and backward LFM images, scanned from left to right and from right to left, respectively, for cells grown at 22 °C. As can be seen from them, most of the yeasts on the LFM images show (34) Colchero, J.; Bielefeldt, H.; Ruf, A.; Hipp, M.; Marti, O.; Mlynek, J. Phys. Status Solidi A 1992, 131, 73. (35) Magonov, S. N.; Whangbo, M.-H. Surface analysis with STM and AFM; VCH: Weinheim, 1996. (36) Overney, R. M.; Meyer, E.; Frommer, J.; Brodbeck, D.; Lu¨thi, R.; Howald, L.; Gu¨ntherodt, H.-J.; Fujihira, M.; Takano, H.; Gotoh, Y. Nature 1992, 359, 133.
circular spots, with a high contrast between forward and backward images, which means that these zones have a surface composition very different than that of their surroundings. Identical images were obtained for cells grown at 37 °C. To detect if these surface inhomogeneities are also related to changes in the adhesion to the hydrophilic AFM tip, force versus distance curves have been collected over the surface. Typical force curves collected for yeasts incubated at 37 °C on the spot region are shown in Figure 3a, and those for yeasts on the rest of the surface are shown in Figure 3b. As is clear from these curves, the spot zone presents, together with a different surface composition, a higher adhesion to the AFM tip. From the force curves taken on different yeasts and on different parts of the two areas of interest, mean values of adhesion force on the spots, Fsp ) 179 ( 47 nN (N ) 70), and on the rest of the surface, Fs ) 39 ( 15 nN (N ) 200), were obtained for the cells grown at 22 °C. Identical behavior was encountered when studying the cells cultured at 37 °C, whose adhesion forces were Fsp ) 135 ( 46 nN (N ) 70) and Fs ) 17 ( 7 nN (N ) 200) on the spots and on the rest of the surface, respectively. Discussion Hydrophobicity has been considered by many authors as a determinant factor mediating the adhesion process.2,3,13,14 The thermodynamic theory predicts that the more hydrophobic yeasts will give more adhesion to hydrophobic substrata and will minimize the adhesion to the hydrophilic ones.17,24 In this context, differences in the adhesion behavior of yeasts to hydrophobic hexadecane and chloroform and hydrophilic silicon nitride tips are expected, based on the changes that culture temperature provokes on the cell surface hydrophobicity.16,37 The analysis of water contact angles (Table 1) shows that C. parapsilosis strain 294 is more hydrophobic when (37) Hazen, K. C.; Hazen, B. W. FEMS Microbiol. Lett. 1993, 15, 83.
Hydrophobicity of Candida parapsilosis 294
it grows at 37 °C and more hydrophilic when it grows at 22 °C, contrary to the relation culture temperaturehydrophobicity described by other authors working with different Candida species, who state that yeasts appear hydrophobic when grown at temperatures below 26 °C and mostly hydrophilic when grown at temperatures around or above 37 °C.8,15,16,37 These hydrophobicity results are well correlated with the changes observed in the kinetic adhesion curves to hexadecane and chloroform at both incubation temperatures (Figure 1) and consequently with the initial deposition rates, which are always higher for cells cultured at 37 °C than at 22 °C for a given solvent. Moreover, there is good correlation among the previous results and the interfacial free energies calculated from water, formamide, and diiodomethane contact angles (Table 2). The values obtained are negative for both solvents and culture temperatures, ranging between -12.9 mJ m-2 for hexadecane on the yeasts cultured at 22 °C and -30.9 mJ m-2 for chloroform on the yeasts incubated at 37 °C. Although these results predict positive adhesion to both solvents, the numerical values do not correlate exactly with the adhesion intensity to each solvent and temperature as it is shown in Figure 1. This discrepancy between thermodynamic predictions and adhesion to the hydrophobic substrata from the MATS technique could be related to the different nature of the techniques employed to evaluate the surface hydrophobicity of yeasts.38 However, another possibility could be considered: because the cell wall is a complex system,39 different answers for each surface patch against each solvent could take place in the adhesion process but could not be reflected in the contact angles measured on lawns of partially dehydrated yeasts, that refer to a characteristic of the yeast surface as a whole. In this context, LFM images (Figure 2) have been collected over the cells in order to test their surface composition homogeneity and force curves have been recorded (Figure 3) over the whole cell surface in order to study the adhesion between the cell surface and the (hydrophilic) silicon nitride tip.40 LFM images (Figure 2) of C. parapsilosis 294 cells reflect the presence of major inhomogeneities in the surface composition, as big circular spots clearly contrasted. These circular spots on the surface of the yeasts have been studied in a previous work41 and seem to be the scar left in the yeast after the formation of a daughter cell. Figure 3 proves the variation over the cell surface of the average adhesion force to the tip. The highly contrasted regions in the LFM images correspond to zones with an average adhesion force value much higher than that of the rest of the surface. These results imply that the intensity of the interaction depends on the part of the surface that interacts with the tip, as it has been suggested that could be happening with hexadecane and chloroform adhesion. Another important issue from the analysis of the adhesion force data, both on the spot and on the rest of the surface, is the higher adhesion force between the tip and yeast for cells cultured at 22 °C than for those incubated at 37 °C, no matter the part selected on the cell surface. This temperature-dependent behavior is well correlated with the lowest adhesion to hexadecane and (38) Pembrey, R. S.; Marshall, K. C.; Schneider, R. P. Appl. Environ. Microbiol. 1999, 65, 2877. (39) Savage, D. C.; Fletcher, M. Bacterial Adhesion, mechanisms and physiological significance; Plenum Press: New York, 1985. (40) Luginb, R.; Szuchmacher, A.; Garrison, M. D.; Lhoest, J. B.; Overney, R. M.; Ratner, B. D. Ultramicroscopy 2000, 82, 171. (41) Me´ndez-Vilas, A.; Gallardo, A. M.; Pe´rez-Giraldo, C.; Gonza´lezMartı´n, M. L.; Nuevo, M. J. Surf. Interface Anal. 2001, 31, 1027.
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chloroform found in the case of cells grown at 22 °C, being mindful of the hydrophobic and hydrophilic character of both solvents and tip, respectively. To show it more clearly, some comparisons have been done between the interaction free energies calculated through contact angles and by the Johnson-Kendall-Roberts (JKR) theory17 assuming it can be applied to our AFM results. To this purpose, the adhesion free energy between the tip and the yeasts has been calculated from24 LW + - + ∆GQY ) -2(xγLW Q γY + xγQγY + xγQγY )
(5)
assuming for silicon nitride the same surface tension, and its components and parameters, as quartz (γQLW ) 31.99, γQAB ) 24.95, γQ+ ) 3.69, γQ- ) 42.14 mJ m-2)42 and using the surface free energy components of the C. parapsilosis strain listed in Table 1. The obtained results are ∆GQY(22 °C) ) -102.10 mJ m-2 and ∆GQY(37 °C) ) -92.26 mJ m-2 for yeasts cultured at 22 and 37 °C, respectively. JKR theory allows one to calculate the interaction free energy between sample and tip from the adhesion force by applying the following expression:
F ) -(3/2)πRW
(6)
where R is the apex radius of the AFM tip, and W is the adhesion work. The adhesion free energy values obtained are ∆Gsp ) -3.79 J m-2 and ∆Gs ) -0.82 J m-2 for cells cultured at 22 °C and ∆Gsp ) -2.86 J m-2 and ∆Gs ) -0.36 J m-2 for yeasts incubated at 37 °C. However, to compare these results with those obtained from macroscopic contact angle measurements, we have calculated a pondered value of the adhesion work taking into account the percentage of the yeast surface area covered by the spot (the average radii of the spot and surface are 0.93 and 3.73 µm, respectively):
∆Gm(22 °C) ) 0.06 ∆Gsp(22 °C) + 0.94 ∆Gs(22 °C) ) -1.00 J m-2 ∆Gm(37 °C) ) 0.06 ∆Gsp(37 °C) + 0.94 ∆Gs(37 °C) ) -0.51 J m-2 Comparison between these results and those of free energy evaluated from the surface free energy and components of the involved phases shows a consistent variation of them with the effect of the growth temperature on the surface properties of yeasts. That is, a less intense interaction of yeast cultured at the higher temperature with hydrophilic substrata, as is indicated by the AFM measurements and ∆GQY evaluation, and the opposite behavior when the interaction deals with hydrophobic substrata (hexadecane and chloroform) as R0 results and ∆GYWH and ∆GYWC calculations prove (Table 2). As far as the general agreement among techniques, it can be interesting to get an idea of the sensitivity of each of them to the changes provoked on the surface properties of yeasts by the culture temperature. However, due to the different magnitude of the results coming from each technique (AFM results are at least 1 order of magnitude higher than those obtained from calculations from contact angle measurements), we have evaluated a normalized (42) Gonza´lez-Martı´n, M. L.; Janczuk, B.; Labajos-Broncano, L.; Bruque, J. M.; Gonza´lez-Garcı´a, C. M. J. Colloid Interface Sci. 2001, 240, 467.
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value of that change as
∆G(37) - ∆G(22) ∆(∆G) | | ) 2| ∆G(37) + ∆G(22) ∆G
P)|
(7)
with ∆G(37) and ∆G(22) being the values of the property evaluated for yeast grown at 37 and 22 °C, respectively. The results obtained are P(AFM, spot) ) 0.28; P(AFM, out of spot) ) 0.78; P(AFM, average surface) ) 0.65; P(interaction surface-hexadecane) ) 0.66; P(interaction surface-chloroform) ) 0.45; P(R0, hexadecane) ) 0.49; P(R0, chloroform) ) 0.31. These results suggest that all of the techniques or methods employed agree in detecting the changes on the microorganism surface wall as a consequence of increasing the yeast growth temperature. Nevertheless, the evaluation of the interaction with hexadecane by R0 or by interfacial free energy from contact angle data has more sensibility to the surface changes than with chloroform. Taking into account that hexadecane and chloroform have identical surface tension components, but hexadecane is
apolar and chloroform is monopolar, and the same techniques were applied with both liquids, it could be suggested that the growth temperature influence is less marked on the surface characteristics related to the acidbase contribution, because of the lower normalized value of the change for chloroform against those for hexadecane with each technique. If so, looking at the normalized values of the change through AFM results, it could be said that the surface composition of the bud scar (P(AFM, spot) ) 0.28) can have an important number of molecules with polar character making the interaction with the tip mainly related to acid-base forces. Acknowledgment. The authors are grateful to the Junta de Extremadura-Consejerı´a de Educacio´n, Ciencia y Tecnologı´a, and the Fondo Social Europeo for the Ph.D. grant awarded to A.M.G.M., for financial support, and for the IPR99C016, IPR00C046, and IPR00A083 projects. We also thank the DGES for the PB97-0378 project and Fondo de Inverstigacio´n Sanitaria, Spain (FIS 00/0293). LA011675Y
