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Attachment of bacteria to model solid surfaces: oligo(ethylene glycol) surfaces inhibit bacterial attachment
ELSEVIER
FEMS Microbiology Letters 142 (1996) 59-63
Attachment of bacteria to model solid surfaces: oligo(ethylene glycol) surfaces inhibit bacterial attachment Linnea K. Ista a, Hongyou
Fan ‘, Oswald Baca b, Gabriel P. Lbpez aj*
a 209 Farris Engineering Center, Department of Chemical and Nuclear Engineering, The University ofNew Mexico. Albuquerque, NM 87131. USA b Department of Biology,The University of New Mexico, Albuquerque, NM 87131, USA
Received 22 May 1996; revised 13 June 1996; accepted 14 June 1996
Abstract Bacterial cell attachment to the surfaces of self-assembled monolayers formed by the adsorption of osubstituted alkanethiols on transparent gold films has been studied under defined bacterial culture and flow conditions. Phase contrast microscopy was used to quantify the attachment of two organisms, one of medical (Staphylococcus epidermidis) and one of marine (Deleya marina) importance. Self-assembled monolayers terminated with hexa(ethylene glycol), methyl, carboxylic acid and fluorocarbon groups were investigated. Over the range of experimental conditions, self-assembled monolayers formed from HS(CH&(0CH&H2)sOH were found to be uniformly resistant to bacterial attachment, with a 99.7% reduction of attachment for both organisms when compared to the most fouled surface for each organism. On other surfaces, S. epidermidis and D. marina were shown to exhibit very different attachment responses to the wettability of the substratum. While the attachment of S. epidermidis correlated positively with surface hydrophilicity, D. marina showed a preference for hydrophobic surfaces. This study suggests that surfaces incorporating high densities of oligo(ethylene glycol) are good candidates for surfaces that interact minimally with bacteria. Keywords: Bacterial attachment; Oligo(ethylene glycol); Self-assembled monolayers; Deleya marina; Staphylococcus epidermidis; Biofouling
1. Introduction The growth
at least attenuate,
of unwanted
living
organisms
on sol-
id, synthetic surfaces exposed to aqueous interfaces is often a problem in industry and in medicine [l]. The attachment of bacterial cells to surfaces is an important component of the biofouling process, and therefore development of ways to prevent, or
* Corresponding author. Tel.: +l (505) 277-4939; Fax: +l (505) 277-5433; E-mail: [email protected] 0378-1097/96/$12.00Copyright
HISO378-1097(96)00243-l
microbial attachment is desirable. Poly(ethylene glycol) (PEG), also referred to as poly(ethylene oxide), has been explored as foulingresistant material [2-4]. Investigators have found that bacteria do not attach well to surfaces composed of PEG-modified polymers [3,4]. Due to the method of preparation of the surfaces used in these initial studies, however, chemical homogeneity of the PEG exposed at the interface often could not be assured, thus the specific contribution of the PEG to the fouling-resistant properties could not be adequately assessed.
0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
L.K. Ista et al, I FEMS Microbiology Letters 142 (1996) 5963
60
A 1 2 3 4
B
--l
Fig. 1. (A) Schematic structure of model surfaces. (I) Self-assembled monolayer of o-substituted alkanethiolates (thickness: - 15-25 A, depending on terminal functional group), (2) gold (thickness: 200 A), (3) chromium (thickness: 10 A), (4) 24x60 mm glass coverslip (thickness: 0.160.19 mm). (B) Parallel flow cell. (1) Aluminum cover, (2) glass coverslip (SAM side down), (3) 0.127 mm Teflon gasket, (4) plexiglass base with stainless steel inlet and outlets. The whole assembly is held together by stainless steel screws (not shown). Drawings not to scale.
Self-assembled monolayers (SAMs) formed by adsorption of o-substituted alkanethiols on gold have been used to study a variety of chemical and physical interfacial phenomena [2]. Recently, methyl and hydroxyl terminated SAMs have been used to study surface energy effects on bacterial adhesion [5]. The surfaces of SAMs are well suited to studies of microbial attachment because they are chemically well-defined, easy to prepare, and the gold substrata can be made optically transparent. They present an advantage over the use of modified commercially available polymers because a wide variety of functional groups can be expressed at the surface with controlled concentration and controlled morphology (i.e. surface roughness). In this report, we present studies of the adhesion
of two microorganisms to SAMs formed from four w-substituted alkanethiols which present surface functional groups that vary widely in their polarity, hydrophobicity, and ionic nature. We were also interested in testing a prevailing paradigm that presents surface tension of the substratum as the primary determinant behind attachment for a given bacterium in a controlled environment. As proposed by Absolom et al. [6], this model states that interactions between bacteria and material surfaces can be predicted from the surface free energies of the bacterium, the bulk medium and the material surface. The present study used bacteria grown in a chemostat, thus cells of the same species should be in the same average metabolic state, and, presumably, their average surface energy will be the same for each experiment. Because the bulk medium is also constant, the only variable should be the surface composition of the SAM. We selected two species of bacteria for this investigation. The first, Staphylococcus epidermidis, has been shown to be involved in sepsis and failure of a number of different medical implants [7]. The second, Deleya marina, a marine bacterium, has many characteristics that make it interesting with regard to the overall understanding of bacterial interactions with surfaces: in addition to the capacity for gliding motility and exopolysaccharide (EPS) production, variants are capable of flagellar motility, and several mutants have been isolated that differ in their surface features and attachment qualities [8].
2. Materials and methods 2.1. Bacterial strains The type strains of Staphylococcus epidermidis (ATCC 14990) and Deleya marina (ATCC 25374) were resuscitated from lyophilized stocks (American Type Culture Collection, Bethesda, MD) and stored at -70°C as a suspension in 20% glycerol in nutrient broth (S. epidermidis) or marine broth 2216 (D. marina). Both were cultured on slants of the appropriate medium for 1 day, single colonies were then inoculated into 5 ml chemostat medium (see below) and grown overnight prior to introduction into the chemostat.
L.K. Ista et al. / FEh4S Microbiology Letters 142 (1996) 59-63
2.2. Growth conditions For both organisms, continuous cultures were established in a 1 liter Virtis continuous culture vessel fed with medium from Pyrex bottles. Flow rate for both organisms was 80 ml/h, giving a dilution rate of 0.16 h-l. Medium for the growth of S. epidermidis was peptone supplemented minimal salts medium with 1 mM glycerol as a carbon source and limiting nutrient [9]. Temperature was maintained at 37°C. Cell density of S. epidermidis grown using this medium and the given dilution rate was 5 X lOa cells/ml. D. marina was grown at room temperature in basal medium with 1 mM glycerol as a carbon source and limiting nutrient (BMG) [lo]. The cell density of this organism in this medium at the given dilution rate was 1.5 X lo8 cells/ml. 2.3. Preparation of self-assembled monolayers SAMs were formed on ethanol-washed coverslips onto which was evaporatively deposited 200 A gold over a base of 10 A chromium (see Fig. 1A). The gold coated slides were rinsed with ethanol and placed in 1 mM solution of the appropriate alkanethiol in ethanol overnight. Four different o-substituted alkanethiols were used. HS(CHs)&H8 was obtained (referred to herein as CH3) Louis, MO. from Aldrich Chemical, St. HS(CH2)&OOH (CF3), HS(CHz)z(CF&CF8 (COOH), and HS(CH2)r1(OCHsCHs)80H (EG6) were synthesized according to published protocols [2,11]. The advancing contact angles of water for the resulting SAMs were: CF3 (120”), CH3 (107”) EG6 (37’) and COOH (< 5”) and agreed well with the published values for these SAMs [2,11]. 2.4. Attachment studies The SAM under consideration was placed into a parallel flow cell with a total volume of 0.097 ml (see Fig. 1B). For experiments with D. marina, 10 ml artificial sea water (ASW: 400 mM NaCl, 100 mM MgS04, 20 mM KCl, CaC12*2H20) [lo] was flushed through the flow cell to wet the substrate before switching to the outflow from the chemostat. A similar procedure was conducted for S. epidermidis with the substitution of phosphate buffered saline (PBS)
61
for ASW. Cells were allowed to attach for 2 h at a flow rate of 1.5 ml/min. Non-attached cells were removed by flushing with 10 ml of ASW or PBS, followed by 20 ml of deionized water to remove salts. The surfaces were evaluated using a Nikon Labophot microscope at a total magnification of 900X with phase contrast. A Student t-test was used to test the significance of differences (P < 0.05) among the mean numbers of attached cells for each bacterium on each surface.
3. Results and discussion The results of the above experiments are summarized in Fig. 2. For both organisms, EG6 attached 99.7% less cells when compared to the most fouled surface for each organism. In our experiments, this reduction is independent of the response of either organism to the wettability of the other substrata. In all cases, the differences between attachment to surfaces were significant by the Student t-test (P-C 0.05). The small numbers of bacteria (4 f 0.9 cells/mm2 for S. epidermidis and 7.4 + 1.4 cells/mm2 for D. marina) which do attach to EG6 may be due to undetected irregularities in our surfaces due to adventitious defects. Different models have been proposed to explain the non-fouling nature of PEG surfaces using both enthalpic and entropic arguments [2]. Favorable enthalpic properties of PEG arise from the high degree of hydration of the ethylene glycol subunits. This property is thought to result in osmotic repulsion of colloid particles and bacteria. Favorable entropic properties of PEG chains are thought to be due to their high excluded volume. Upon binding of a particle, freedom of movement is restricted, leading to a decreased entropy in the system. The resulting increase in enthalpy and decrease in entropy would lead one to predict that attachment of a particle to surfaces composed of PEG would not be thermodynamically favored. The attachment of S. epidermidis to the surfaces other than EG6 shows results similar to those obtained in previous studies [12], in that the adherence of this organism is directly related to the hydrophilicity of the substratum. Since the surface wettability of S. epidermidis does not change significantly from
L.K. Ista et al. I FEMS Microbiology Letters 142 (1996) 59-63
62
CF3
CH3
EG6
COOH
Surface
B 3000 “E E 2500 2 3
2000
P r: I
Iso
0 CF3
CH3
EG6
COOH
Surface Fig. 2. Attachment of bacteria to SAMs formed of o-substituted alkanethiolates on gold. (A) Staphylococcus epidermidis (ATCC 14990). (B) Deleya marina (ATCC 25374). For all surfaces, duration of attachment was 2 h at a flow rate of 1.5 mUmin. Each data point represents the average f standard error of 10 samples. For each sample, 68 fields of view were averaged. Surfaces are arranged in order of increasing hydrophilicity.
(ATCC 25374). Although this organism attaches more readily in our studies to hydrophobic surfaces than hydrophilic surfaces, no clear trend is seen between the degree of hydrophobicity and attachment. These results differ from previous studies, which indicated that this strain adhered more strongly to hydrophilic than to hydrophobic surfaces [14]. The previous study noted that EPS deficient mutants prefer hydrophobic surfaces. While the previous study used rich medium (W-broth: 1% tryptone and 0.5% yeast extract in ASW), our medium was more minimal and may not have supported EPS production. A qualitative test for EPS based on the method of Christensen [I 51 was conducted on 48 h D. marina cultures grown in different media in both glass and polystyrene culture tubes. Large amounts of EPS were detected as stained material following the addition of 0.2% saffranin to emptied tubes in which cells had been grown in W-broth and marine broth 2216, whereas none was detected for BMG (data not shown). Observations of colony morphology and aggregation patterns for BMG-grown cells also suggest that no EPS was produced. Our results indicate that SAMs composed of oligo(ethylene glycol)-terminated alkanethiolates resist attachment of D. marina and S. epidermidis. Because these two organisms differ in phylogeny and natural environment, these results suggest that the non-fouling properties of PEGS may be applicable to most, if not all, environmentally encountered bacteria. It is also clear from this investigation that the wettability of a surface is only one factor in determining whether bacteria will adhere to it. The observation that EG6 does not follow trends of adherence predicted by its relative surface hydrophilicity, along with the results obtained for D. marina, indicates that other physicochemical factors may be involved. Our studies in the immediate future will focus on the physicochemical interactions between bacteria and a variety of SAMs with different surface characteristics.
Acknowledgments strain to strain and is hydrophilic [13], this result is predicted from the model proposed by Absolom et al. [6]. This relationship is not observed for D. marina
We thank Ms. Erika Johnston, Mr. Tommy Fong and Mr. Lewis Worley for technical assistance. This research was supported by ONR Grants N00014-95-
L. K. Ista et al. I FEMS
l-0255, NOOO14-95-1-0901 and N00014-95-1315, NSF Grant HRD-9450473.
Microbiology
Letters 142 (1996)
and
[9]
References [l] Cooksey, K.E. and Wigglesworth-Cooksey, B. (1992) The design of antifouling surfaces: Background and some approaches. In: Biofilms - Science and Technology (Melo, L.F., Bott, T.R., Fletcher, M. and Capdeville, B., Eds.) pp. 529. Kluwer Academic, Dordrecht. [2] Prime, K.L. and Whitesides, G.M. (1993) Adsorption of proteins onto surfaces containing end-attached oligo(ethylene oxide): a model system using self-assembled monolayers. J. Am. Chem. Sot. 115, 1071410721. [3] Desai, N.P., Hossainy, F.A. and Hubbell, J.A. (1992) Surfaceimmobilized polyethylene oxide for bacterial repellence. Biomaterials 13, 417420. [4] Blainey, B.L. and Marshall, K.C. (1991) The use of copolymers to inhibit bacterial adhesion and biofilm formation on hydrophobic surfaces in marine habitats. Biofouling 4, 309318. [5] Wiencek, K.M. and Fletcher, M. (1995) Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers. J. Bacterial. 177, 1959-1966. [6] Absolom, D.R., Lamberti, F.V., Policova, Z., Zingg, W., VanOss, C.J. and Neumann, A.W. (1983) Surface thermodynamics of bacterial adhesion. Appl. Environ. Microbial. 46, 90-99. [7] Wilcox, M.H. (1993) Medical-device associated adhesion. In Microbial Biofilms: Formation and Control (Denyer, S.P., Gorman, S.P. and Sussman, M., Eds.), pp. 131-146. Blackwell Scientific, Oxford. [8] Shea, C., Lovelace, L.J., and Smith-Sommersville, H.E. (1995)
[lo]
[l l]
[12]
[13]
[14]
[15]
59-63
63
Deleya marina as a model organism for studies of bacterial colonization and biofilm formation. J. Ind. Microbial. 15, 290-296. Evans, E., Brown, M.R.W. and Gilbert, P. (1994) Iron chelator, exopolysaccharide and protease production in Staphylococcus epidermidis: a comparative study of the effects of specific growth-rate in biofilm and continuous culture. Microbiology 140, 153-157. Kersters K. (1992) The genus Deleya. In: The Prokaryotes (Balows, A., Trtiper, H.G., Dworkin, M, Harder, W. and Schliefer, K.-H., Eds.), pp. 3189-3197. Springer-Verlag. Berlin. Bain, C.D., Troughton, E.B., Tao, Y.-T., Evall, J., Whitesides, G.M. and Nuzzo, R.G. (1989) Formation of monolayer films by spontaneous assembly of organic thiols from solution onto gold. J. Am. Chem. Sot. 111, 323-335. Ferreiros, C.M., Carballo, J., Criado, M.T., Sainz, V. and de1 Rio M.C. (1989) Surface free energy and interaction of Sraphylococcus epidermidis with biomaterials. FEMS Microbial. Lett. 60, 89-94. Hogt, A.H., Dankbert, J., Hulstaert, C.E. and Feijen, J. (1986) Cell surface characteristics of coagulase-neeative staphylococci and their adherence to fluorinated poly(ethylenepropylene). Infect. Immun. 51, 294301. Shea; C., Nunley, J.W., Williamson, J.C. and Smith-Sommersville, H.E. (1991) Comparison of the adhesion properties of Deleya marina and the exopolysaccharide-defective mutant strain DMR. Appl. Environ. Microbial. 33, 3107-3113. Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F, Melton, D.M. and Beachey, E.H. (1985) Adherence of coagulase-negative staphylococci to plastic tissue culture plates a quantitative model for the adherence of staphylococci to medical devices. J. Clin. Microbial. 22, 996 1006.
FEMS Microbiology Letters 142 (1996) 59-63
Attachment of bacteria to model solid surfaces: oligo(ethylene glycol) surfaces inhibit bacterial attachment Linnea K. Ista a, Hongyou
Fan ‘, Oswald Baca b, Gabriel P. Lbpez aj*
a 209 Farris Engineering Center, Department of Chemical and Nuclear Engineering, The University ofNew Mexico. Albuquerque, NM 87131. USA b Department of Biology,The University of New Mexico, Albuquerque, NM 87131, USA
Received 22 May 1996; revised 13 June 1996; accepted 14 June 1996
Abstract Bacterial cell attachment to the surfaces of self-assembled monolayers formed by the adsorption of osubstituted alkanethiols on transparent gold films has been studied under defined bacterial culture and flow conditions. Phase contrast microscopy was used to quantify the attachment of two organisms, one of medical (Staphylococcus epidermidis) and one of marine (Deleya marina) importance. Self-assembled monolayers terminated with hexa(ethylene glycol), methyl, carboxylic acid and fluorocarbon groups were investigated. Over the range of experimental conditions, self-assembled monolayers formed from HS(CH&(0CH&H2)sOH were found to be uniformly resistant to bacterial attachment, with a 99.7% reduction of attachment for both organisms when compared to the most fouled surface for each organism. On other surfaces, S. epidermidis and D. marina were shown to exhibit very different attachment responses to the wettability of the substratum. While the attachment of S. epidermidis correlated positively with surface hydrophilicity, D. marina showed a preference for hydrophobic surfaces. This study suggests that surfaces incorporating high densities of oligo(ethylene glycol) are good candidates for surfaces that interact minimally with bacteria. Keywords: Bacterial attachment; Oligo(ethylene glycol); Self-assembled monolayers; Deleya marina; Staphylococcus epidermidis; Biofouling
1. Introduction The growth
at least attenuate,
of unwanted
living
organisms
on sol-
id, synthetic surfaces exposed to aqueous interfaces is often a problem in industry and in medicine [l]. The attachment of bacterial cells to surfaces is an important component of the biofouling process, and therefore development of ways to prevent, or
* Corresponding author. Tel.: +l (505) 277-4939; Fax: +l (505) 277-5433; E-mail: [email protected] 0378-1097/96/$12.00Copyright
HISO378-1097(96)00243-l
microbial attachment is desirable. Poly(ethylene glycol) (PEG), also referred to as poly(ethylene oxide), has been explored as foulingresistant material [2-4]. Investigators have found that bacteria do not attach well to surfaces composed of PEG-modified polymers [3,4]. Due to the method of preparation of the surfaces used in these initial studies, however, chemical homogeneity of the PEG exposed at the interface often could not be assured, thus the specific contribution of the PEG to the fouling-resistant properties could not be adequately assessed.
0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
L.K. Ista et al, I FEMS Microbiology Letters 142 (1996) 5963
60
A 1 2 3 4
B
--l
Fig. 1. (A) Schematic structure of model surfaces. (I) Self-assembled monolayer of o-substituted alkanethiolates (thickness: - 15-25 A, depending on terminal functional group), (2) gold (thickness: 200 A), (3) chromium (thickness: 10 A), (4) 24x60 mm glass coverslip (thickness: 0.160.19 mm). (B) Parallel flow cell. (1) Aluminum cover, (2) glass coverslip (SAM side down), (3) 0.127 mm Teflon gasket, (4) plexiglass base with stainless steel inlet and outlets. The whole assembly is held together by stainless steel screws (not shown). Drawings not to scale.
Self-assembled monolayers (SAMs) formed by adsorption of o-substituted alkanethiols on gold have been used to study a variety of chemical and physical interfacial phenomena [2]. Recently, methyl and hydroxyl terminated SAMs have been used to study surface energy effects on bacterial adhesion [5]. The surfaces of SAMs are well suited to studies of microbial attachment because they are chemically well-defined, easy to prepare, and the gold substrata can be made optically transparent. They present an advantage over the use of modified commercially available polymers because a wide variety of functional groups can be expressed at the surface with controlled concentration and controlled morphology (i.e. surface roughness). In this report, we present studies of the adhesion
of two microorganisms to SAMs formed from four w-substituted alkanethiols which present surface functional groups that vary widely in their polarity, hydrophobicity, and ionic nature. We were also interested in testing a prevailing paradigm that presents surface tension of the substratum as the primary determinant behind attachment for a given bacterium in a controlled environment. As proposed by Absolom et al. [6], this model states that interactions between bacteria and material surfaces can be predicted from the surface free energies of the bacterium, the bulk medium and the material surface. The present study used bacteria grown in a chemostat, thus cells of the same species should be in the same average metabolic state, and, presumably, their average surface energy will be the same for each experiment. Because the bulk medium is also constant, the only variable should be the surface composition of the SAM. We selected two species of bacteria for this investigation. The first, Staphylococcus epidermidis, has been shown to be involved in sepsis and failure of a number of different medical implants [7]. The second, Deleya marina, a marine bacterium, has many characteristics that make it interesting with regard to the overall understanding of bacterial interactions with surfaces: in addition to the capacity for gliding motility and exopolysaccharide (EPS) production, variants are capable of flagellar motility, and several mutants have been isolated that differ in their surface features and attachment qualities [8].
2. Materials and methods 2.1. Bacterial strains The type strains of Staphylococcus epidermidis (ATCC 14990) and Deleya marina (ATCC 25374) were resuscitated from lyophilized stocks (American Type Culture Collection, Bethesda, MD) and stored at -70°C as a suspension in 20% glycerol in nutrient broth (S. epidermidis) or marine broth 2216 (D. marina). Both were cultured on slants of the appropriate medium for 1 day, single colonies were then inoculated into 5 ml chemostat medium (see below) and grown overnight prior to introduction into the chemostat.
L.K. Ista et al. / FEh4S Microbiology Letters 142 (1996) 59-63
2.2. Growth conditions For both organisms, continuous cultures were established in a 1 liter Virtis continuous culture vessel fed with medium from Pyrex bottles. Flow rate for both organisms was 80 ml/h, giving a dilution rate of 0.16 h-l. Medium for the growth of S. epidermidis was peptone supplemented minimal salts medium with 1 mM glycerol as a carbon source and limiting nutrient [9]. Temperature was maintained at 37°C. Cell density of S. epidermidis grown using this medium and the given dilution rate was 5 X lOa cells/ml. D. marina was grown at room temperature in basal medium with 1 mM glycerol as a carbon source and limiting nutrient (BMG) [lo]. The cell density of this organism in this medium at the given dilution rate was 1.5 X lo8 cells/ml. 2.3. Preparation of self-assembled monolayers SAMs were formed on ethanol-washed coverslips onto which was evaporatively deposited 200 A gold over a base of 10 A chromium (see Fig. 1A). The gold coated slides were rinsed with ethanol and placed in 1 mM solution of the appropriate alkanethiol in ethanol overnight. Four different o-substituted alkanethiols were used. HS(CHs)&H8 was obtained (referred to herein as CH3) Louis, MO. from Aldrich Chemical, St. HS(CH2)&OOH (CF3), HS(CHz)z(CF&CF8 (COOH), and HS(CH2)r1(OCHsCHs)80H (EG6) were synthesized according to published protocols [2,11]. The advancing contact angles of water for the resulting SAMs were: CF3 (120”), CH3 (107”) EG6 (37’) and COOH (< 5”) and agreed well with the published values for these SAMs [2,11]. 2.4. Attachment studies The SAM under consideration was placed into a parallel flow cell with a total volume of 0.097 ml (see Fig. 1B). For experiments with D. marina, 10 ml artificial sea water (ASW: 400 mM NaCl, 100 mM MgS04, 20 mM KCl, CaC12*2H20) [lo] was flushed through the flow cell to wet the substrate before switching to the outflow from the chemostat. A similar procedure was conducted for S. epidermidis with the substitution of phosphate buffered saline (PBS)
61
for ASW. Cells were allowed to attach for 2 h at a flow rate of 1.5 ml/min. Non-attached cells were removed by flushing with 10 ml of ASW or PBS, followed by 20 ml of deionized water to remove salts. The surfaces were evaluated using a Nikon Labophot microscope at a total magnification of 900X with phase contrast. A Student t-test was used to test the significance of differences (P < 0.05) among the mean numbers of attached cells for each bacterium on each surface.
3. Results and discussion The results of the above experiments are summarized in Fig. 2. For both organisms, EG6 attached 99.7% less cells when compared to the most fouled surface for each organism. In our experiments, this reduction is independent of the response of either organism to the wettability of the other substrata. In all cases, the differences between attachment to surfaces were significant by the Student t-test (P-C 0.05). The small numbers of bacteria (4 f 0.9 cells/mm2 for S. epidermidis and 7.4 + 1.4 cells/mm2 for D. marina) which do attach to EG6 may be due to undetected irregularities in our surfaces due to adventitious defects. Different models have been proposed to explain the non-fouling nature of PEG surfaces using both enthalpic and entropic arguments [2]. Favorable enthalpic properties of PEG arise from the high degree of hydration of the ethylene glycol subunits. This property is thought to result in osmotic repulsion of colloid particles and bacteria. Favorable entropic properties of PEG chains are thought to be due to their high excluded volume. Upon binding of a particle, freedom of movement is restricted, leading to a decreased entropy in the system. The resulting increase in enthalpy and decrease in entropy would lead one to predict that attachment of a particle to surfaces composed of PEG would not be thermodynamically favored. The attachment of S. epidermidis to the surfaces other than EG6 shows results similar to those obtained in previous studies [12], in that the adherence of this organism is directly related to the hydrophilicity of the substratum. Since the surface wettability of S. epidermidis does not change significantly from
L.K. Ista et al. I FEMS Microbiology Letters 142 (1996) 59-63
62
CF3
CH3
EG6
COOH
Surface
B 3000 “E E 2500 2 3
2000
P r: I
Iso
0 CF3
CH3
EG6
COOH
Surface Fig. 2. Attachment of bacteria to SAMs formed of o-substituted alkanethiolates on gold. (A) Staphylococcus epidermidis (ATCC 14990). (B) Deleya marina (ATCC 25374). For all surfaces, duration of attachment was 2 h at a flow rate of 1.5 mUmin. Each data point represents the average f standard error of 10 samples. For each sample, 68 fields of view were averaged. Surfaces are arranged in order of increasing hydrophilicity.
(ATCC 25374). Although this organism attaches more readily in our studies to hydrophobic surfaces than hydrophilic surfaces, no clear trend is seen between the degree of hydrophobicity and attachment. These results differ from previous studies, which indicated that this strain adhered more strongly to hydrophilic than to hydrophobic surfaces [14]. The previous study noted that EPS deficient mutants prefer hydrophobic surfaces. While the previous study used rich medium (W-broth: 1% tryptone and 0.5% yeast extract in ASW), our medium was more minimal and may not have supported EPS production. A qualitative test for EPS based on the method of Christensen [I 51 was conducted on 48 h D. marina cultures grown in different media in both glass and polystyrene culture tubes. Large amounts of EPS were detected as stained material following the addition of 0.2% saffranin to emptied tubes in which cells had been grown in W-broth and marine broth 2216, whereas none was detected for BMG (data not shown). Observations of colony morphology and aggregation patterns for BMG-grown cells also suggest that no EPS was produced. Our results indicate that SAMs composed of oligo(ethylene glycol)-terminated alkanethiolates resist attachment of D. marina and S. epidermidis. Because these two organisms differ in phylogeny and natural environment, these results suggest that the non-fouling properties of PEGS may be applicable to most, if not all, environmentally encountered bacteria. It is also clear from this investigation that the wettability of a surface is only one factor in determining whether bacteria will adhere to it. The observation that EG6 does not follow trends of adherence predicted by its relative surface hydrophilicity, along with the results obtained for D. marina, indicates that other physicochemical factors may be involved. Our studies in the immediate future will focus on the physicochemical interactions between bacteria and a variety of SAMs with different surface characteristics.
Acknowledgments strain to strain and is hydrophilic [13], this result is predicted from the model proposed by Absolom et al. [6]. This relationship is not observed for D. marina
We thank Ms. Erika Johnston, Mr. Tommy Fong and Mr. Lewis Worley for technical assistance. This research was supported by ONR Grants N00014-95-
L. K. Ista et al. I FEMS
l-0255, NOOO14-95-1-0901 and N00014-95-1315, NSF Grant HRD-9450473.
Microbiology
Letters 142 (1996)
and
[9]
References [l] Cooksey, K.E. and Wigglesworth-Cooksey, B. (1992) The design of antifouling surfaces: Background and some approaches. In: Biofilms - Science and Technology (Melo, L.F., Bott, T.R., Fletcher, M. and Capdeville, B., Eds.) pp. 529. Kluwer Academic, Dordrecht. [2] Prime, K.L. and Whitesides, G.M. (1993) Adsorption of proteins onto surfaces containing end-attached oligo(ethylene oxide): a model system using self-assembled monolayers. J. Am. Chem. Sot. 115, 1071410721. [3] Desai, N.P., Hossainy, F.A. and Hubbell, J.A. (1992) Surfaceimmobilized polyethylene oxide for bacterial repellence. Biomaterials 13, 417420. [4] Blainey, B.L. and Marshall, K.C. (1991) The use of copolymers to inhibit bacterial adhesion and biofilm formation on hydrophobic surfaces in marine habitats. Biofouling 4, 309318. [5] Wiencek, K.M. and Fletcher, M. (1995) Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers. J. Bacterial. 177, 1959-1966. [6] Absolom, D.R., Lamberti, F.V., Policova, Z., Zingg, W., VanOss, C.J. and Neumann, A.W. (1983) Surface thermodynamics of bacterial adhesion. Appl. Environ. Microbial. 46, 90-99. [7] Wilcox, M.H. (1993) Medical-device associated adhesion. In Microbial Biofilms: Formation and Control (Denyer, S.P., Gorman, S.P. and Sussman, M., Eds.), pp. 131-146. Blackwell Scientific, Oxford. [8] Shea, C., Lovelace, L.J., and Smith-Sommersville, H.E. (1995)
[lo]
[l l]
[12]
[13]
[14]
[15]
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