Attachment of staphylococci to various synthetic polymers

Zbl. Bakt. Hyg. A 256,479-489 (1984)

Attachment of Staphylococci to Various Synthetic Polymers Anlagerung von Staphylokokken an verschiedene synthetische Polymere A. LUDWICKA!, B. }ANSEN 2 , T. WADSTROM 3, and G. PULVERER 1 Institute of Hygiene, University of Cologne, 5000 Cologne 41, Fed. Rep. of Germany Institute of Physical Chemistry, University of Cologne, 5000 Cologne 41, Fed. Rep. of Germany 3 Department of Bacteriology and Epizootology, University of Agricultural Sciences, Uppsala, Sweden 1

2

With 4 Figures· Received September 20, 1983 . Accepted September 26, 1983

Abstract Attachment of staphylococci to different synthetic polymers used for medical purposes was studied in applying the bioluminescent technique. The number of attached bacterial cells was determined by measuring the light emission resulting from the reaction between firefly luciferase and ATP present in adhered staphylococcal cells. It was shown that staphylococci attach to synthetic polymers within a few minutes, although one hour incubation is required to reach a constant maximum value of attached cells. Ten different synthetic polymers and five Staphyloccocus epidermidis strains were investigated in our study. The relationship between surface properties of polymers and bacterial attachment was studied. Various physicochemical parameters of synthetic polymers and bacteria were determined (contact angle, surface tension). It was demonstrated that bacterial attachment decreases with decreasing contact angle and with increasing surface tension of synthetic materials. Modifications of surface charge and hydrophobicity of solid materials were also investigated. It could be proved that especially negatively charged and hydrophilic synthetic polymers show very decreased staphylococcal attachment.

Zusammenfassung In der vorliegenden Arbeit wurde die Anlagerung von Staphylokokken an verschiedene synthethische, medizinisch bedeutsame Polymere untersucht. Mit Hilfe der Bioluminiszenztechnik wurde die Zahl der angelagerten Staphylokokkenzellen bestimmt. Die uberpriiften Staphylokokkenstamme adharierten an die synthetischen Polymere innerhalb weniger Minuten, 1 Stunde Inkubation wurde jedoch zum Erreichen eines konstanten Maximums an angelagerten Staphylokokken benotigt, Unsere Studie umfafste 10 verschiedene synthetische Polymere und 5 Staphylococcus epidermidis-Stiunme. Getestet wurden insbesondere Wech-

480

A.Ludwicka, B.]ansen, T.Wadstrom, and G.Pulverer

seiwirkungen zwischen bestimmten Oberflacheneigenschaften der Kunststoffe und der Staphylokokken-Anlagerung, uns interessierten hierbei besonders Oberflachenspannung und Kontaktwinkel. Wir konnten zeigen, daiS die Staphylokokken-Anlagerung mit abnehmendem Kontaktwinkel und mit zunehmender Oberflachenspannung des Polymers abnimmt. Modifikation der Oberflachenladung und der Hydrophobizitat der Polymere wurden auf ihre Auswirkung auf die Staphylokokken-Anlagerung gepriift. Besonders negativ geiadene und hydrophile Kunststoffe zeigten eine stark herabgesetzte Staphylokokken-Adharenz,

Introduction Adhesion of cells to solid substrates has been the subject of many investigations (3, 5,6, 7, 8, 10, 14, 17,28). Most prior studies on bioadhesion phenomena have dealt with the influence of substrate surface properties on the relative adherence and growth of mammalian cells (1, 3). Because of blood compatibility and infection problems the examination of biomedical materials is of considerable clinical interest. Besides thrombosis, speticemia is one of the most frequent complications in using intravenous catheters (15, 16,23,24,26,27). The attachment of bacteria to solid surface like biomaterials is an important phenomenon because of its possible role as the very first step in the development of an infection. Several parameters like critical surface tension, surface free energy, charge and hydrophobicity, both of the substrates and the attached cells, are surely involved in this phenomenon (3, 7, 8,10,14,25). A relationship between the surface free energy of various synthetic materials and the biological response (e. g. cell adhesion or fibrous encapsulation) was postulated (3, 14), but still the mechanisms of the interaction between bacterial cells and synthetic substrates is not yet understood. In the following paper the bacterial attachment of Staphylococcus epidermidis strains to various synthetic polymers is investigated. S. epidermidis strains were chosen because they are the most common organisms causing infections of indwelling artificial devices, particularly prosthetic heart valves and intravenous catheters (23, 24, 26, 27). The influence of surface properties of pure polymers and of surface-modified polymers on the attachment behaviour is examined.

Material and Methods Bacterial strains and culture conditions Five Staphylococcus epidermidis strains classified according to Kloos and Schleifer (13), from the culture collection from the Institute of Hygiene, University of Cologne, were studied: Three of these strains (KH 6, KH 11 and KH 12) were isolated from infected intravenous catheters, the two other strains (Gloor 1/1 and Gloor 99) were cultivated from skin infections. The strains were cultured in nutrient broth (Difco) in Erlenmeyer flasks for 18 h at Bacteria were harvested by centrifugation (2500 X g, 15 min. 20°C) and washed three times in phosphate buffered saline (PBS, 0.13 M NaC!, 0.02 M phosphate buffer, pH 7.4). Cells were resuspended in PBS and suspensions were filtered through a Millipore membrane (pore size 8 urn) to remove clumps of bacteria. The absorption degree of suspension was measured using a Beckman spectrophotometer (A 600 nm) and compared with the results of colony counting on blood agar. The staphylococcal suspensions were then adjusted to 10 8 cells per ml. Fresh bacterial suspensions were prepared every day.

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Attachment of Staphylococci

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Preparation of synthetic polymer surfaces The following synthetic polymers were examined in our study: Silicone rubber (Silastic''), (Dow Corning Corp., Midland/Mich., USA), polyetherurethane WHS (Biosearch Inc., Raritan, N. J. USA), polypropylene (amorphous), polyethylene (Hostalen''), and poly(ethylene)-eterephtalate (Hostaphan"), (Fa. Kalle, Wiesbaden, FRG), polyvinylidene fluoride (PVF2 ) (Solef"), (Fa. Alkor, Miinchen, FRG), and cellulose acetate (Ultraphan''), (Fa. LonzaWerke, Weil am Rhein, FRG). Small discs of the polymers (d = 6 mm) were cleaned with a detergent and ethanol followed by extensive rinsing in distilled water. They were sterilized by irradiation in using a 60Co-y-source (activity 5000 Ci) at a dose of 2.5 Mrad. Polymer pieces prepared in this way were used in all experiments.

Modification of surface charge and hydrophobicity of some synthetic polymers Surface modified polyetherurethans were grafted with HEMA-a hydrophilic monomeraccording to the method of Jansen and Ellinghorst (11). PVF2 was modified by grafting with acrylic acid and thereafter treated with KOH. Polyethylene was coated with lysosyme (Serva), which is an hydrophilic and basic protein, and with "crude slime" preparation-a hydrophilic and acidic substance isolated from S. epidermidis strains KH 11 (non published data). Polyethylene pieces were incubated with 1 % solution of lysosyme and "slime" in PBS, pH 7.4, overnight at 28°C. After incubation polyethylene samples were washed with 10 ml PBS, the excess of PBS was removed by air drying. Such prepared samples were used for attachment studies and the contact angels of coated polyethylene was measured.

Contact angle measurements Contact angles on synthetic polymers and on bacteria were performed using a contact angle goniometer of Fa. Lorentzen and Wetters (Stockholm) combined with a special cell (Rame-Hart, New Jersey). Triple distilled water (for polymers) and physiological saline solution (for bacteria) were taken as contact angle liquids. The polymer samples were cleaned with a detergent, ethanol and triple distilled water prior to the measurements. The angles of 10 sessile drops on each sample were measured and the mean average taken as a final result. Contact angels on bacteria were determined according to a method described by Van Oss (22). Flat layers of staphylococci were obtained by pouring washed bacterial cells (10 10 cells/ml) on agar-corvered microscopic slides, followed by air drying for 15 min. All samples were stored in an atmosphere of definite humidity before the measurements. Again, a set of 10 measurements were done with each sample.

Hydrophobic interaction chromatography (HIC) Pasteur pipettes were plugged with glass wool, packed with Octyl-Sepharose (Pharmacia) and equilibrated with 1 M ammonium sulfate in 0.1 M sodium phosphate, pH 7.0. After washing, the bacterial suspension (100f.l1, approx. 5 X 10 9 cells/ml) was applied to a gel bed and eluted with the same buffer. The absorbance (A600 nm) of the eluate was measured and the percentage of bacteria absorbed to octyl-sepharose gel was calculated. The strains which absorbed to the gel more than 75 % were considered as hydrophobic (12).

Measurement of bacterial attachment to synthetic polymers by a bioluminescent assay (17)

Instrumentation: The light emitted in the firefly assay of ATP was measured at room temperature using a Luminometer 1250 (LKB-Wallac Turku, Finland). The signal was recorded with a Digital display unit with printer (LKB-Wallac) 1250-101. Reagents: One vial of ATP-Monitoring Reagent (LKB-Wallac) was reconstituted with 50 ml of tris-EDTA buffer (0.1 M tris and 2 mM EDTA adjusted to pH 7.75 with acetic acid). 31 Zbl. Bakt. Hyg. A 256

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One vial of ATP Standard (LKB-Wallac) was reconstituted with 10 ml of tris-EDTA. buffer to a final ATP concentration of 101lM. Reagents were kept at room temperature during the assay. Synthetic polymer pieces were incubated with 250lll of staphylococcal suspensions (108 cells/ml) at 20°C. After one hour incubation the polymer pieces were rinsed with 10 ml PBS to remove non- or loosely adhered bacterial cells. The number of cells adhering to polymers was determined by the luminescent technique. After removing the excess of PBS from polymers they were transfered to cuvettes containing 50111 of 2.5 % trichloracetic acid (TCA) to extract bacterial ATP. Complete and rapid mixing was assured by immediately shaking the cuvette. The amount of bacterial ATP in the extract was determined after addition of 1 ml of reconstituted ATP Monitoring Reagent and calculated from ratio III stand. multiplied by the amount of ATP in 20111 of added ATP Standard (0.2 nmoles), (18, 19). From this the blank (calculated in the same way) was subtracted to obtain the final result. The number of attached cells per crrr' was calculated from standard curves prepared with known amounts of bacterial cells in the suspension. Mean values of three samples of each polymer were taken as a final result. Results Attachment of different S. epidermidis strains to various synthetic materials was investigated using the bioluminescence technique (17). Fig. 1 shows the kinetics of staphylococcal attachment to polyethylene: bacterial adherence occurs within a few minutes, after about one hour of incubation time a plateau value is reached. Table 1 shows the results of the attachment of all five S. epidermidis strains to several synthetic polymers and glass. Bacterial adhesion is expressed as number of attached bacteria per cm 2 of polymer after 1 h of incubation. Furthermore, the water contact angles and surface properties like surface free energy (y SV) and polar contribution to surface free energy (y f) of the solid substrate are listed. The polymers in the table are divided into three groups: the first one contains commercially available unmodified polymers used as basic materials for medical applications. The second group contains two surface modified polyetherurethanes (grafted with HEMA, a hydrophilic monomer, according

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484

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to a method described in (14) with increased surface hydrophilicity). In the third group surface modified PVF2 (grafted with acrylic acid and treated with KOH) and glass are included; these last two substances exhibit an extreme hydrophilicity and have a negatively charged surface whereas all other polymers are noncharged. Attachment of the S. epidermidisstrains KH 11, KH 12 and Gloor 1/1 to the neutral, unmodified polymers (with water contact angles ranging from 109 to 58°C) appears to be variable, but in most cases values of more than 104 attached bacteria per cm2 are found. These polymers are either hydrophobic with no polar groups or with moderate polar contributions to their surface free energy. Attachment to modified polyetherurethans with higher values of polar contribution seems to be less, and attachment to very hydrophilic and negatively charged modified PVF2 and glass is generaly very low. Fig. 2. shows the results for the strains KH 11, KH 12 and Gloor 1/1 in dependence from the surface energy y SV of the solids used. Although not all data fit in well, a curve

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Fig.2. Attachment of three hydrophobic staphylococcalstrains to severalsolid substrates vs. surface free energy (y SV) of the substrate. A~A S. epidermidis strain KH 11, . - . S. epidermidisstrain KH 12, e-. S. epidermidisstrain Gloor 111. Number of bacteria in the incubation mieture 108 cells/mI. can be drawn showing a decrease of attachment with increasing y SV and thus increasing hydrophilicity. To emphasize this, the polar contribution to surface free energy y SV of the solid substrate (either from literature data or from own measurements) was plotted against the number of attached bacteria (Fig. 3). The majority of the points lies on a curve, showing decreased attachment to those solids with high ability for polar interactions. The other two S. epidermidis strains KH 6 and Gloor 99 do not cause such great differences in attachment to various materials, especially in the case of KH 6 strain the bacterial amount is nearly on one level (exceptions are modified PVF2 and glass which show again very low adhesion (Table 1.)). Determination of the hydrophobicity of the bacterial strains (performed with contact angle and HIe measurements) revealed that KH 6 and Gloor 99 strains are much more hydrophilic than strains KH

Attachment of Staphylococci

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11, KH 12 and Gloor 1/1. In this case attachment seems to be independent from y SV of the solid substrate (Fig. 4.). As can be seen in Table 2, a significant inhibition of attachment of an hydrophobic and hydrophilic strain was observed when polyethylene was coated with lysosyme and especially with an acidic slime substance.

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486

A.Ludwicka, B.]ansen, T.Wadstrom, and G.Pulverer Table 2. Attachment of S. epidermidis strains to polyethylene coated with Iysosyme and crude slime preparation Polyethylene coated with

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Discussion There exist several types of interactions which participate in the attachment process of cells to cellular or solid substrates. Besides specific bindings, where adhesive parts on the surface of bacteria (adhesins) as well as on host cell membranes (receptors) are involved (4), nonspecific factors influencing the bacterial adherence also occur (3). These factors are important determinants in the formation of multiple bounds between bacteria and host cells or solid substrates. The net charge and hydrophobicity of the surface of both bacterial cells and host cells or solid surfaces create repulsive forces between the cells or substrates (3, 14). Marshall et a1. (21) reported that adhesion of marine bacterial specimens to solid, smooth surfaces is a spontaneous adsorptive phenomenon, where electrostatic forces and a dispersion balance between cells and the substrate in aqueous media is involved. These authors also suggested that the initial stage of bacterial attachment based on the theory of lyophobic colloid stability is reversible. A more permanent adhesion was observed when extracellular polymers interact between bacteria and the surface (21). Most of the reports concerning attachment to solid substrates deal with marine bacterial specimens (6, 7, 21), whereas vety little is known about the attachement of staphylococci to synthetic polymers. This problem is of great importance because coagulase-negative staphylococci are very often involved in infections of prosthetic implants or medical devices (2, 16,23,24,26,27, 28). Our investigations on the relationship between surface properties of unmodified and modified synthetic polymers and staphylococcal attachment showed (Table 1 and Fig. 2.) that the adherence of hydrophobic strains of S. epidermidis is dependent on the surface free energy y SV of the substrate. A decrease of attachment with increase of y SV and thus increasing hydrophilicity was observed. Our results are in general accordance with the data of Fletcher (7), Hogt et al. (10) and of Absolom et a1. (1), who developed a thermodynamic model for the adhesion of cells to solid surface. In their considerations, adhesion is governed by the charge in free energy of adhesion (ll Fadh ) and is dependent on the surface free energy of the suspending liquid medium (y LV) and that of the solid substrate (y SV) and of bacteria (y BV). They distinguished between three possibilities: 1.) y BV< y LV: in this case II padh increases with increasing y SV of the solid

Attachment of Staphylococci

487

substrates, attachment should increase with increasing hydrophobicity (or decrease with increasing hydrophilicity), 2.) y BV > YLV: 6pdh decreases with increasing y SV and adhesion should decrease with increasing hydrophobicity, 3.) y BV = YLV: then 6pdsh = 0, adhesion should be independent of the surface free energy of the solid substrate. In our experiments, y LV (saline solution, PBS, values of 67 mN . m") is greater than the surface free energy of bacteria and thus adhesion should decrease with increasing y SV. As can be seen from Fig. 2., our results are in good agreement with the thermodynamic model. The attachment of more hydrophilic strains (KH 6 and Gloor 99) to all synthetic polymers (Fig. 4.) does not show such relationship to surface free energy as the hydrophobic strains do. In this case surface free energy y SV is higher and approaches the value of y LV for the suspending liquid medium (PBS or saline solution). As mentioned above for such a case, adhesion should be independent from y SV of the solid substrate, and this is nearly true for our results (Fig. 4.). Bacterial adhesion to very hydrophilic PVFrg-AAc and glass is very low in all experiments. A reason for this might be the fact that both solids have also a negative surface charge. A significant decrease of bacterial attachment was also observed when polyethylene was coated with lysosyme or with crude slime preparation isolated from strain KH 11 (Table 2). Both substances covered polyethylene homogenously changing extremely their surface hydrophilicity (contact angles from 95 to 0°). The inhibition of bacterial attachment to polyethylene coated with slime - a strongly acidic carbohydrate polymer - was higher in comparison to lysosyme (basic protein) coated polyethylene suggesting that electrostatic forces playa significant role in attachment processes. Inhibition of attachment caused by slime substance seems to be nonspecific and mostly due to the charge of molecule, therefore the adherence of hydrophobic and hydrophilic strains was inhibited to the same extent. As most of the bacteria have also a negative surface charge, a repulsion of the bacteria from the solid substrate due to electrostatic forces must be assumed. From the data presented here it may be concluded that the interaction of staphylococcal strains with synthetic polymers is a multifactorial event. Both hydrophobic and hydrophilic interactions as well as eletrostatic forces seem to mediate the mechanisms of attachment, which in our opinion cannot be regulated by a single factor. Besides the forces found in this study other factors, not yet identified, may be existing and interfering with the complex mechanisms of attachment. Acknowledgements. We would like to thank Prof. G. Uhlenbruck for his critical reading of the manuscript and helpful suggestions. The authors A. Ludwicka and B. Jansen acknowledgethe granting of an Alexander von Humboldt-Fellowship and Fritz Thyssen-Fellowship respectively.

References 1. Absolom, D. R., A. W. Neumann, W. Zingg, and C. J. van Oss: Thermodynamicstudies of cellular adhesion. Trans. Amer. Soc. Artif. Int. Organs XXV (1979), 152-158 2. Archer, G. L.: Antimicrobial susceptibility and selection of resistance among Stapbylo-

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coccus epidermidis isolates recovered from patients with infections of indwelling foreign devices. Antimicrob. Agents Chemother. 14 (1978), 353-359 3. Baier, R. E.: Comments on cell adhesion to biomaterial surfaces: Conflicts and concerns. J. Biomed. Mat. Res. 16 (1982) 173-175 4. Beachey, E. H.: Bacterial adherence: Adhesin-receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. infect. Dis. 143 (1981) 325-345 5. Christensen, G. D., W. A. Simpson, A. 1. Bisno, and E. H. Beachey: Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infect. Immun. 37 (1982) 318-326 6. Corpe, W. A.: Attachment of marine bacteria to solid surfaces. In: Adhesion in biological systems, R. S. Manly (ed.), p. 73-87. Academic. Press, New York (1970) 7. Fletcher, M. and G. 1. Loeb: Influence of substratum characteristics on the attachment of a marine Pseudomonad to solid surfaces. Appl. Environ. Microbiol. 37 (1979) 67-72 8. Gerson, D. F. and D. Scheer: Cell surface energy, contact angles and phase partition. III. Adhesion of bacterial cells to hydrophobic surfaces. Biochem. Biophys. Acta 602 (1980) 506-510 9. Gibbson, R. J. and J. Van Houte: Bacterial adherence in oral microbial ecology. Ann. Rev. Microbiol. 29 (1975) 19-44 10. Hogt, A. H., J. Feijen, J. Dankert, and J. A. De Vries: Adhesion of Staphylococcus epdermidis and Staphylococcus saprophyticus onto FEP-teflon and cellulose acetate. International Conference on Biomed. Polymers, July 1982, Durham/G. B., p. 39-47 11. Jansen, B. and G. Ellinghorst: Radiation initiated grafting of hydrophilic and reactive monomers on polyetherurethane for biomedical application. Radiat. Phys. Chern. 18 (1981) 1195-1202 12. Jonsson, P., and T. Wadstrom: High surface hydrophobicity of Staphylococcus aureus as revealed by hydrophobic interaction chromatography. Curro Microbiol. 8 (1983) in press 13. Kloos, W. E. and K. H. Schleifer: The genus Staphylococcus. In: M. Starr et al. (eds.), Prokaryotes, p. 1548-1569. Springer-Verlag, Berlin (1982) 14. Lerche, D.: The adhesiveness to the cell surface with regard to its electric and steric strucutre. Bioelectrochem. Bioenerg. 8 (1981) 293-299 15. Locci, R., G. Peters, and G. Pulverer: Microbial colonization of prosthetic devices. I. Microtopographical characteristics of intravenous catheters as detected by Scanning Electron Microscopy. Zbl. Bakt. Hyg., I. Abt. Orig. B 173 (1981) 285-292 16. Locci, R., G. Peters, and G. Pulverer: Microbial colonization of prosthetic devices. III. Adhesion of staphyloccoci to lumina of intravenous catheters perfused with bacterial suspensions. Zbl. Bakt. Hyg., I. Abt. Orig. B 173 (1981) 300-307 17. Ludwicka, A., R. Locci, B. Jansen, G. Peters, and G. Pulverer: Microbial colonization of prosthetic devices. V. Attachment of coagulase-negative staphylococci and "slime"production on chemically pure synthetic polymers. Zbl. Bakt. Hyg., I. Abt, Orig. B 177 (1983) 527-532 18. Ludwicka, A., 1. M. Switalski, A. Lundin, G. Pulverer, and T. Wadstrom: Measurements of bacterial attachment to a solid surface (synthetic polymers) by a bioluminescent assay. Appl. Bact. (submitted for publication) 19. Lundin, A.: Application of firefly luciferase. In: M. Serio and M. Pazzagli (eds.), In: luminescent assay: Perspectives in endocrinology and clinical chemistry, p. 29-45. Raven Press, New York (1982) 20. Lundin, A. and A. Thore: Comparison of method for extraction of bacterial adenine nucleotides determined by firefly assay. Appl. Microbiol. 30 (1975) 713-721 21. Marshall, K. c., R. Stout, and R. Mitchell: Mechanism of the initial events in sorption of maxime bacteria surfaces. J. gen. Microbiol. 68 (1971) 337-348 22. Van Oss, and C. F. Gillmann: Phagocytosis as a surface phenomenon. J. Reticulendothel. Soc. 12 (1972) 283-292 23. Peters, G., R. Locci, and G. Pulverer: Microbial colonization of prosthetic devices. II.

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Scanning electron microscopy of naturally infected intravenous catheters. Zbl. Bakt. Hyg., I. Abt. Orig. B 173 (1981) 293-299 Peters, G., R. Locci, and G. Pulverer: Adherence and growth of coagulase-negative Staphylococci on surface of intravenous catheters. J. infect. Dis. 146 (1982) 479-482 Schrader, J. E.: On adhesion of biological substances to low energy and solid surfaces. J. Colloid Interface Sci. 88 (1982) 288-297 Sewell, C. M., J. E. Clarridge, E. J. Young, and R. K. Guthrie: Clinical significance of coagulase-negative staphylococci. J. Clin. Microbiol. 16 (1982) 236-239 Speller, D. C. E., and G. Mitchell: Coagulase-negative staphylococci causing endocarditis after cardiac surgery. J. Clin. Path. 26 (1973) 517-522 Surgarman, G.: In vitro adherence of bacteria to prosthetic vascular grafts. Infection 10 (1982) 9-12

Prof. Dr. G. Pulverer, Hygiene-Institut d. Universitat, Goldenfelsstr. 19-21, D-5000 K6ln