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Teichoic acid enhances adhesion of Staphylococcus epidermidis to immobilized fibronectin
Microbial Pathogenesis 2001; 31: 261–270
Article available online at http://www.idealibrary.com on
doi:10.1006/mpat.2001.0469
MICROBIAL PATHOGENESIS
Teichoic acid enhances adhesion of Staphylococcus epidermidis to immobilized fibronectin Muzaffar Hussain∗, Christine Heilmann, Georg Peters & Mathias Herrmann† Institute of Medical Microbiology, University Hospital of Muenster, Germany (Received June 11, 2001; accepted in revised form August 30, 2001)
Adhesion is a prerequisite for coagulase-negative staphylococci to cause invasive disease and may be mediated by adhesive host molecules adsorbed on implanted polymers. In this study, we can confirm previous observations demonstrating binding of Staphylococcus epidermidis to fibronectin (FN) adsorbed polymer surfaces. So far, the nature of FN-recognizing adhesin(s) in S. epidermidis remains elusive. Since teichoic acids (TA) have been shown to exert binding functions for extracellular matrix molecules in several Gram-positive species, we have purified wall TA of S. epidermidis laboratory strains KH11 and RP62A, as well as clinical isolate AB9. Using a polymethylmethacrylate (PMMA) coverslip adhesion assay, a microtitre plate assay and a particle agglutination assay, we found that purified TA significantly enhanced adhesion of S. epidermidis KH11 and RP62A to FN coated surfaces. Enhanced adhesion was dose-dependent and saturable. Preincubation, either of microorganisms or of FN coated surfaces, with TA promoted adhesion, while adhesion to TA-adsorbed PMMA was comparably low. This observation may suggest a potential role of cell wall carbohydrates as bridging molecules between microorganisms and immobilized FN in early steps of S. epidermidis pathogenesis. 2001 Academic Press Key words: Staphylococcus epidermidis, bacterial adhesion, fibronectin, teichnoic acid.
Introduction Staphylococcus epidermidis and other coagulasenegative staphylococcal species, previously † Present address: Institute of Medical Microbiology and Hygiene, University of the Saarland Medical School, Bldg. #43, Homburg 66421, Germany. ∗ Author for correspondence. E-mail: muzaffa@uni-muenster. de 0882–4010/01/120261+10 $35.00/0
considered harmless commensals, are now recognized as the most important pathogens for infections of implanted prosthetic material and catheter-associated blood stream infection. The events associated with initiation and course of these infections have been intensely studied, and distinctive mechanisms contributing to adhesion of microorganisms to the uncoated polymer [1, 2], proliferation and accumulation on the surface resulting in biofilm formation [3–6], and persistence of the sessile bacterial population until 2001 Academic Press
262 0.7
(a)
Adhesion (%)
0.6 0.5 0.4 0.3 0.2 0.1 0.0
1.0
KH11
RP62A
AD22
AB7
AB9
RP62A
AD22
AB7
AB9
(b)
0.9 0.8 0.7 OD460 nm
removal of the polymer material [7, 8] have been identified. Generally, it is thought that S. epidermidis prosthetic device infections occur through colonization of naked polymer surfaces by microorganisms resident in the surgical or the catheter wound [9]. However, in addition to indwelling device infection, S. epidermidis are also capable of causing invasive disease, such as osteomyelitis [10], deep tissue infection [11] or native valve endocarditis [12]. Therefore, in these entities as well as in certain types of polymer-associated infection (e.g., tunnel infections of implanted catheters or ventricular assist device drive lines), S. epidermidis may bind to immobilized host factors rather than to unadsorbed plastic [13]. In vitro studies have demonstrated that S. epidermidis adheres to polymeradsorbed fibronectin (FN), yet, in contrast to the FN-binding proteins in S. aureus [14], a FNbinding adhesin in S. epidermidis has not been identified. Teichoic acid (TA), an essential wall constituent of staphylococci, has been implicated in the binding of S. epidermidis binding fibrin clots [15] and in the adhesion of S. aureus to epithelial cells [16]. Therefore, the goal of this study was to evaluate a potential role of S. epidermidis TA as a candidate molecule mediating binding of S. epidermidis to immobilized FN.
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0.6 0.5 0.4 0.3 0.2 0.1 0.0
KH11
Figure 1. Promotion of adhesion of five strains of S. epidermidis to FN-adsorbed surfaces. (a) PMMA coverslips (b) microtitre plate wells. Binding to BSA (Φ) used as control and to FN (Ε). Experimental conditions described in Materials and Methods.
Results Interaction of S. epidermidis with soluble and immobilized FN (i) Coagglutination test. S. epidermidis bound soluble FN as demonstrated in a coagglutination reaction using formalin fixed and heat killed Staphylococcus aureus Cowan 1 cells sensitized with anti-FN-Abs. All five tested S. epidermidis isolates revealed a positive agglutination reaction, yet agglutination occurred to a different extent with strains KH11 and AD22 yielding a (+++), strains RP62A and AB7 a (++) and strain AB9 a (+) reaction. (ii) Particle agglutination test. Using FN coated latex particles results similar to those of coagglutination test results were obtained. Two strains, KH11 and AD22, showed strong agglutination reaction (+++), two strains, RP62A and AB7, agglutinated moderately (++) and a fifth strain, AB9, agglutinated weakly (+). (iii) Adhesion to
FN coated PMMA cover slips. Pre-adsorption of FN to PMMA promoted adhesion of the five S. epidermidis isolates tested in this study [Fig. 1(a)]. (iv) Adhesion to FN coated microtitre plate. Promotion in adhesion of these isolates to FNadsorbed surfaces was also observed when using a polystyrene microtitre plate adhesion assay [Fig. 1(b)]. Adhesion of strain KH11 to FN coated polystyrene surfaces was found to be dosedependent with concentrations of FN 5–50 g/ ml (data not shown).
Effect of treatment of bacteria with trypsin and periodate oxidation on adhesion This experiment was performed to characterize the nature of the receptor for FN on staphylococcal cells surface. In separate experiments bacterial cell surface proteins were removed by
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Table 1. Adhesion of S. epidermidis to FN-adsorbed PMMA or FN-adsorbed polystyrene and effect of soluble TA Radiometric adhesion assay
KH11 RP62A AD22 AB7 AB9
Microtitre plate assay
Albumin FN FN plus TA Percent adhesion (mean, SD)
Albumin
0.03 0.09 0.04 0.06 0.09
0.09 0.08 0.04 0.08 0.05
(0.01) (0.07) (0.02) (0.05) (0.06)
0.51 0.57 0.65 0.27 0.39
(0.05) (0.06) (0.02) (0.03) (0.01)
1.74 1.74 1.59 0.74 0.45
(0.11) (0.12) (0.16) (0.10) (0.08)
trypsination and carbohydrates were removed by periodate oxidation. The adhesion reaction was found to be both trypsin and periodatesensitive, because pre-treatment of bacteria with these reagents caused a reduction of KH11 adhesion to FN-PMMA by 65 and 74%, and of RP62A by 61 and 63%, respectively. The effect of trypsin suggested a possible role of bacterial surface proteins in the interaction with immobilized FN, adhesion assays were performed using a mutant defective in expression of AtlE. AtlE is an autolysin of strain S. epidermidis O47 with vitronectin (VN) binding activity in Western-ligand assays [1], and the AtlE analogue from strain S. epidermidis strain AB9 recognized both FN and VN [17]. Adhesion of the mutant to FN-PMMA was slightly elevated (mean adhesion, 0.86%) compared to the wild-type strain (0.34%), but this was accompanied by slightly enhanced adhesion to BSA-PMMA (0.11 vs 0.06%). Thus, AtlE does not appear to mediate adhesion of S. epidermidis O-47 to FN-adsorbed surfaces.
(0.02) (0.50) (0.03) (0.03) (0.04)
FN FN plus TA OD492nm (mean, SD) 0.22 0.53 0.57 0.10 0.32
(0.06) (0.04) (0.13) (0.01) (0.04)
0.54 0.79 0.93 0.42 0.56
(0.08) (0.09) (0.04) (0.03) (0.10)
p-anisidine or AgNO3/NaOH. In another chromatogram, a glycerol–phosphate spot was disclosed with ammonium molybdate+perchloric acid+HCl mixture. The chromatogram stained with ninhydrin showed only one spot close to the alanine marker. Hydrolysates prepared from isolated TA from S. epidermidis RP62A, KH11 and AB9 did exhibit these characteristics typical for staphylococcal wall TA [18, 19]. (ii) Enzymatic and chemical analysis. In TA from strain RP62A, 165 g of glucose and 40 g of glucosamine were determined per mg of freezedried material. Taking molecular masses of polymer units of glucose and glucosamine as 162 and 203, respectively, then these data suggests presence of 1 M of glucose and 0.2 M of glucosamine in each mg of freeze-dried material. (iii) Reaction with lectins. TA isolated from three strains (RP62A, KH11 and AB9) showed strong precipitation reactions with lectins concanavalin-A and lectin from Triticum vulgaris (wheat germ lectin) specific for glucose and glucosamine respectively. The reaction with lectins suggests presence of glucose and glucosamine as glucosy substituents.
Purification and analysis of TA (i) Thin layer chromatography. Cell walls isolated from the middle layer of the sucrose gradient were hydrolysed in 6 M HCl for 18 h at 105°C, and amino acids were separated by thin layer chromatography. Only alanine, glycine, glutamic acid and lysine were detected with ninhydrin, a clear indication of a pure cell wall preparation. TA extracted from purified cell walls by either procedure was hydrolysed in 3 M HCl for 3 h and analysed by thin layer chromatography. Spots having Rglucose values close to those of glucose, glucosamine and glycerol–phosphate were identified with
Role of TA in the FN–S. epidermidis interaction These findings prompted further evaluation of the role of cell wall TA to the binding reaction. Upon addition of TA purified by anion exchange chromatography (final concentration, 16.5 g/ ml), an increase in adhesion of all five tested isolates, both in the radiometric adhesion assay as well as in the microtitre plate assay, were observed with strain AB9 being least promoted (Table 1). Using the particle agglutination assay, addition of TA purified from either RP62A, AB9
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M. Hussain et al. (a) 2.0
***
3.0 (b) 2.5
***
2.0 Adhesion (%)
Adhesion (%)
1.5
1.0
1.5 1.0
0.5 0.5
0.0
KH11
RP62A
0.0
KH11
Figure 2. (a) Effect of different TA preparations on promotion of S. epidermidis adhesion to FN-adsorbed PMMA. Untreated (Φ) or FN-pre-adsorbed (25 g in 1 ml PBS, 60 min, 37°C) (Ε Φ, Ε, and Ε Φ) PMMA coverslips were incubated with 4×106 cfu/ml of [3H]-thymidine-labelled bacteria in 1 ml of Ca++/Mg++PBS supplemented with 0.5% human serum albumin (60 min, 37°C, shaking water bath), then washed. The number of adherent microorganisms was expressed as percentage of inoculum. In some experiments, suspensions containing radiolabelled bacteria and FN-PMMA were supplemented with TA purified from the DEAE cellulose column (Ε) or from the concanavalin-A column (Ε Φ) (16.5 g/ml, respectively) prepared as described in Materials and Methods. Shown are means of three determinations±SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) and subsequent Student-Newman-Keuls test for determination of the significance levels of differences between observations. ANOVA, P<0.001; ∗∗∗, P<0.01 of FN alone vs DEAE- or concavalin-A-eluate supplement. (b) Effect of two TA preparations on adhesion of S. epidermidis KH11 to FN-PMMA. TA was prepared either from S. epidermidis KH11 (Ε) or from RP62A (Ε Φ) and used to supplement the adhesion buffer as described in Fig. 1.
or KH11 to FN coated beads resulted in a moderate agglutination (+/++), yet, when FNbeads were first treated with purified TA and then incubated with washed cells of strains KH11, RP62A or AB9, an immediate and strong (+++) agglutination reaction was detected. Control (BSA-adsorbed) beads revealed no agglutination whether incubated with TA alone or with TA plus bacterial cells. Strains KH11 and RP62A were further studied. Lectin-affinity chromatography did not enhance the biologic activity of purified TA, since TA-enhanced adhesion was similar using either purification procedure [Fig. 2(a)]. No discernible difference in adhesion was observed when TA isolated upon either TA extraction procedure (trichloroacetic acid or lysostaphin–lysozyme extraction) was used (not shown). For subsequent experiments, TA was prepared from digestion of cell walls with lysostaphin–lysozyme and purified by anion-exchange chromatography. The promotion of adhesion by TA was not isolate-dependent, i.e. TA purified from strain RP62A promoted adhesion of strain KH11 to a similar
extent when compared with TA from strain KH11 [Fig. 2(b)]. Adhesion was found to be dosedependent with concentrations of >16.5 g/ml, resulting in saturation of TA-mediated adhesion enhancement (Fig. 3). While these experiments demonstrated the adhesion-promoting effect of TA in co-incubation with FN-PMMA and staphylococci (Fig. 4), this effect was also to be observed, albeit to a lesser extent, if PMMA coverslips were preincubated with TA then rinsed and tested for adhesion. Moreover, the extent of adhesion of bacteria pre-adsorbed with TA then washed was found to be almost identical to adhesion to FN-PMMA in the presence of TA. In contrast, microorganisms adhered to PMMA pre-adsorbed with TA only to a small extent.
Interaction of TA with DIG-labelled ligands DIG labelled FN bound very well to TA and DIG labelled FG bound to a lesser extent to immobilized TA on a nitrocellulose membrane, while DIG labelled VN did not show any binding to TA (data not shown).
Teichoic acid and S. epidermidis adhesion
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addition of oxidized TA are: strain KH11 OD492nm, 0.24±0.05 (mean±SD, n=3), 0.25 (0.08); strain AB9 0.36 (0.07), 0.35 (0.05).
1.4
Adhesion (%)
1.2 1.0 0.8
Effect of soluble FN on adhesion to TA coated polystyrene surface
0.6 0.4 0.2 1.65
16.5 Teichoic acid added (µg/ml)
165
Figure 3. Adhesion promotion by TA as a function of TA concentration. Various concentrations of TA were supplemented to the adhesion buffer as described in Fig. 1, and adhesion of radiolabelled S. epidermidis KH11 was subsequently determined. Shown are means of triplicate determinations±SD.
***
When a bacterial suspension containing 50 g/ ml FN was added to a microtitre plate wells precoated with TA, a slightly enhanced adhesion of strain KH11 and AB9 was observed: strain KH11, OD492nm, 0.09±0.03 (mean±SD, n=3) enhanced to 0.14 (0.05) and strain AB9, 0.07 (0.04) enhanced to 0.12 (0.05). The possible explanation includes: (i) a small amount of immobilized TA on a polystyrene surface or (ii) the fact that functional binding sites of TA may not be any more available for bridging between cell surface components and soluble FN after immobilization of TA.
***
Adhesion (%)
2.0
Discussion
1.5
1.0
0.5
0.0
KH11
RP62A
Figure 4. Effect of coincubation vs preincubation of TA either with FN-PMMA or with bacteria. Control assays either without FN (Φ) or with FN-PMMA alone (Ε Φ) were performed as described in Fig. 1. Alternatively, TA (16.5 g/ml) was either added during the adhesion incubation (Ε), or either FN-PMMA (Ε Φ) or radiolabelled bacteria (P) were preincubated with TA, then washed and incubated for bacterial adhesion. In some experiments, PMMA coverslips without FN were incubated in the presence of TA (16.5 g/ml) and radiolabelled bacteria (Q). Shown are means±SD of three determinations. ANOVA, P<0.001, ∗∗∗, P<0.01 of FN-PMMA alone vs coincubation or preincubation with TA.
Effect of oxidized TA on adhesion The oxidized TA failed to enhance adhesion of strains KH11 and AB9 to polystyrene microtitre plate coated with FN. Values before and after
In this study, we describe the interaction of S. epidermidis with FN and identify cell wall TA as an adhesive molecule recognizing adsorbed FN. After contact with blood, plasma proteins are sequentially adsorbed to polymeric surfaces, with high-abundant, low molecular-weight proteins, such as albumin, being rapidly and preferentially adsorbed but subsequently replaced by less abundant, more surface active proteins such as FN and fibrinogen [20]. These observations are in good agreement with data obtained from ex vivo human [21] and canine [22] catheter materials witnessing deposition of significant amounts of adhesive molecules on the polymer substrates. Accordingly, various studies on the role of FN on S. epidermidis adhesion, albeit with contrasting results, have been reported [21, 23–29], while adhesion to other immobilized matrix proteins was found to show more interstrain variability or to be low [23]. Experimental conditions contributing to differences in physicochemical binding forces, notably in surface hydrophobicity, may account at least for a part of these differences [1, 26, 30]. To model the physiologic conditions addressed in this study, we have performed our assays in a physiologic, i.e. albumin-containing milieu, and by using three different types of assays we
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have confirmed that polymer-adsorbed FN does indeed promote adhesion of S. epidermidis to the substrate. Recently, we have reported on putative S. epidermidis adhesins recognizing FN in Westernligand blots [17]. While one of the released molecules was ornithine carbamoyltransferase, an intracellular enzyme not involved in adhesion to surfaces, here we present evidence that another S. epidermidis protein recognizing VN and FN in Western-ligand assays, the S. epidermidis autolysin AtlE, does not contribute to adhesion to FN. Thus, a proteinaceous adhesin recognizing FN remains unidentified in S. epidermidis. Gram-positive cocci have been shown to interact with whole cells or extracellular matrix protein, notably FN, via TA and/or lipoteichoic acid. In numerous studies {reviewed in [31]}, streptococcal lipoteichoic acid (LTA) has been suggested to bind to FN and these observations have prompted a model of streptococcal attachment to extracellular matrix or eukaryotic cells involving a two-step mechanism including LTA and proteinaceous receptors [32]. This model has been challenged but provides a basis for further research in multiple-step adhesion mechanisms of Gram-positive cocci including staphylococci [15, 16, 33]. In general, research on the role of cell wall TA as adhesins is hampered by the fact that mutants of staphylococci deficient in TA are not available, since wall TA appear to be essential for survival. Our evidence suggesting a role of TA as an S. epidermidis adhesin for FN is based on the following observations: (i) the demonstration of dose-dependency and saturability of TAenhanced adhesion of S. epidermidis to adsorbed FN, (ii) the fact that S. epidermidis adheres only slightly to TA-pre-adsorbed PMMA in the absence of FN, and (iii) the effect of carbohydratemodifying periodate on adhesion. As protease treatment also reduced adhesion, both proteinand carbohydrate-type ligands may be involved, however, it has been demonstrated for streptococci that trypsin digestion of bacteria removes most of the TA from cell surface [34]. Our findings suggest that TA forms a bridge between the FN molecule and the S. epidermidis cell surface. This may occur via rebinding of TA through its anionic backbone to cationic molecules on cell surface [35], however, additional research, e.g. using mutants defective in TA-modifying enzymes, is necessary to further determine the molecular mechanisms involved. Further work
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is also necessary to explore the potential of these findings for novel prophylactic or therapeutic strategies directed against S. epidermidis infections.
Materials and Methods Bacterial strains and culture media S. epidermidis isolates KH11 [36] and RP62A (ATCC 35984) were used. In addition, clinical S. epidermidis blood stream isolates AD22, AB7 and AB9 were selected from the strain collection of our institute for further study. For determination of the role of AtlE on adhesion to FN, in some experiments wild-type strain O-47 and its AtlEdeficient mutant, mut1 [1], was also used. Identification of clinical isolates as S. epidermidis on a species level was performed using the ID 32 biochemical test kit (BioMerieux, Marcy-l’Etoile, France). Working cultures were maintained on blood agar plates (for strain mut1 supplemented with erythromycin 10 g/ml), and fresh overnight cultures were grown on brain–heart broth/ agar (Merck, Darmstadt, Germany) and Muller Hinton broth/agar (Mast, Merseyside, U.K.).
Solid phase adhesion assay For radiomateric analysis of S. epidermidis adhesion to coated solid surfaces, a previously described assay was used [37]. Briefly, solution containing 25 g/ml human plasma FN (Chemicon, Temecula, CA, U.S.A.) was allowed to adsorb to PMMA coverslips for 60 min at 37°C. Thereafter, coverslips were washed with PBS and incubated in a shaking water bath with [3H] thymidine-labelled S. epidermidis cells [4×106 cfu in 1 ml PBS containing 0.5% bovine serum albumin (Sigma-Aldrich, Deisenhofen, Germany)] for 60 min at 37°C. After adhesion, the PMMA coverslips were washed with PBS three times, and adherent cpm counts were determined. In some experiments TA was added as g of glucose per ml of adhesion assay.
Microtitre plate adhesion assay Polystyrene plates (96 well) (Greiner, Frickenhausen, Germany) were coated with FN (50 g/ml−1 in 50 mM sodium carbonate buffer,
Teichoic acid and S. epidermidis adhesion
pH 9.6) for 18 h at 4°C, then blocked with 3% BSA in TBS (25 mM Tris–HCl, 100 mM NaCl, pH 7.5) and washed. Bacteria were grown for 18 h at 37°C, washed once with TBS, adjusted to an OD578nm 1.0 in TBS, and to each well 200 l of bacterial suspension was added. Plates were incubated at 37°C for 1 h, then wells were washed three times, stained with safranin for 1 min, and the plate was read at 492 nm on a Titertek-multiscan-8 (Flow laboratories, Bonn, Germany). To study the effect of soluble FN on adhesion to TA coated polystyrene surface, the above detailed experiment was performed with the following two modifications: (i) the surface was coated with TA (16.5 g/ml) and (ii) the bacterial suspension contained 50 g/ml FN.
Coagglutination test The preparation of coagglutination reagent (SAC+anti-FN-Abs) involved fixing of protein A found on surface of S. aureus cowan1 (ATCC 12598) cells with formaldehyde and then killing bacteria by heating [38, 39]. To the resulting protein A solid phase, anti-FN-Abs (Dako) were coupled. Bacteria were grown in BHI for 18 h at 37°C with shaking, washed twice with PBS and incubated at room temperature after suspension in PBS containing 50 g/ml FN. After 1 h, FN treated bacteria were washed twice with PBS and resuspended in PBS to OD598nm of 1.0. For the coagglutination test, 10 l of FN exposed bacterial suspension was mixed with 20 l of coagglutination reagent on a microscope slide. After 3 min reactions were scored as negative (−) or from weakly positive (+) to strongly positive (+++). No agglutination was found when the PBS used to suspend bacteria and coagglutination reagent was tested as a control.
Particle agglutination assay Method of Naidu et al. [40] was used. Briefly, 200 l of blue latex beads (Sigma) were preadsorbed with FN (100 g/ml) or with BSA for control in sodium carbonate buffer (pH 9.5), then washed. Bacteria were grown overnight in BHI, washed, and resuspended in PBS. FN-latex beads (20 l) were mixed with 20 l of bacterial suspension, and after 3 min the agglutination reaction was scored as negative (−), or from weak positive (+) to strong positive (+++).
267
Preparation of cell walls For isolation of cell wall, bacteria were grown in 800 ml TSB in two 1 l flasks, each inoculated with 0.5 ml of an 18 h culture in the same medium and incubated with shaking at 37°C for 18 h. Bacteria were pelleted by centrifugation (10 000×g, 10 min, 4°C) and washed twice with distilled water. Washed bacteria were broken by the method of Huff et al. [41], shaking with glass beads in a Braun cell homogenizer (Braun, Melsungen, Germany). For the separation of walls by sucrose gradient centrifugation, the method of Yoshida et al. [42] was used. Cell walls from the middle layer of sucrose gradient were resuspended in 50 mM Tris–HCl pH 7.8 plus trypsin 250 g/ml, and kept at 37°C with shaking for 4 h. The suspension was centrifuged (32 000×g, 30 min, 4°C), sedimented walls were washed with 1 M NaCl, then six times with distilled water, and freeze-dried. For characterization, freeze-dried cell walls (5 mg) in 6 M HCl (1 ml) in a sealed glass tube were heated at 105°C in an oven for 18 h. Acid hydrolysate was dried in a dessicator over silica gel in presence of NaOH under vacuum. Acid from hydrolysate was completely removed by suspending the dried hydrolysate in a few drops of water and repeating the drying step at least three times. Amino acids were separated by thin layer chromatography and detected by ninhydrin spray.
Isolation of TA from cell walls TA was extracted from cell walls by two methods: (i) digestion with lysozyme+ lysostaphin. Cell walls (1 mg/ml) were suspended in 50 mM Tris–HCl, pH 8, containing 1.2 mM EDTA (Sigma) and 0.145 M NaCl (Merck, Darmstadt, Germany). The suspension was first digested with a mixture of recombinant lysostaphin (Applied Micro Inc., New York, U.S.A.) and lysozyme (Merck) for 3 h at 37°C, followed by 1 mg/ml trypsin (Sigma) digestion for 4 h at 37°C. After enzyme treatment, the suspension was centrifuged (30 000×g, 4°C, 30 min), then passed through a 0.22 m membrane filter (Millipore Corporation, Bedford, U.S.A.) to remove cell wall fragments. Five volume of ethanol was added to the filtrate. After overnight equilibration at 4°C, the liquid was discarded and the precipitate containing TA was freeze-dried. (ii) Extraction with trichloroacetic
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acid (TCA) by the method of Archibald et al. [43].
Anion-exchange and affinity chromatography TA was fractionated by ion-exchange chromatography on DEAE-cellulose and affinity chromatography as described previously for the fractionation of extracellular products [19, 44]. (i) Briefly, the DEAE cellulose (Sigma) column (1.5×11 cm) was equilibrated with 0.1 M ammonium acetate (Merck) buffer (pH 5.5), and 25 mg of crude TA in 2 ml buffer was loaded onto the column. The column was eluted stepwise with increasing concentrations of NaCl in buffer. Fractions were analysed by a phenol/ sulfuric acid assay [45] and an assay for total phosphorus [46]. Fractions making peaks were pooled, dialysed against distilled water for 24 h, and freeze-dried. (ii) Affinity chromatography. Material eluted with 0.5 M NaCl in 50 mM ammonium acetate from DEAE-cellulose was loaded on a concanavalin A sepharose 4-B column (Sigma) equilibrated with 0.1 M sodium acetate (pH 6.0) containing 1 M NaCl, 1 mM of each CaCl2, MgCl2, MnCl2 and 0.02% NaN3. The column was washed with 20 bed volumes of loading buffer, then bound material was eluted with five bed volumes of 0.1 M methyl -Dglucopyranoside in loading buffer, and assayed for phosphorus. The eluted material was dialysed against distilled water then freeze-dried.
Analysis of TA by thin layer chromatography TA was hydrolysed in 3 M HCl for 3 h at 100°C in a boiling water bath. Acid from hydrolysate was removed as detailed earlier for cell wall hydrolysis. Prepared hydrolysate was separated on thin layer chromatography plates and detected using standard methods [47, 48].
Estimation of glucose and glucosamine in TA preparation Glucose concentration was determined by a highly specific hexokinase method of Carroll et al. [49] using glucose kit 115-A (Sigma). For measurement of glucosamine, method of Levvy
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and McAllan [50] with glucosamine. HCl (Sigma) as standard was used.
Reaction with lectins Two lectins, concanavalin-A (Sigma) and lectin from Triticum vulgaris (wheat germ lectin; Sigma), were used. TA suspension (20 l) was added to lectin solution (10 mg/ml, 20 l) on a glass slide. Results were recorded as negative or positive (clumping) after 1 min.
Interaction of TA with FN TA was immobilized on nitrocellulose membrane (Schleicher & Scuell, Dasel, Germany) by allowing a 5 l TA solution to penetrate the membrane and subsequently dried. The remaining binding sites on the membrane were blocked by 3% BSA (fraction V, Sigma), then separate membranes were probed with DIGlabelled FN, FG or VN for 1 h. The membranes were washed three times with TBS-T and transferred to a solution of alkaline phosphataseconjugated anti-DIG-Abs (Roche, Mannheim, Germany) for 1 h. Spots binding FN, FG or VN were detected in an alkaline phosphatase colour reaction (Bio-Rad) in accordance with the instructions of the supplier.
Removal of surface proteins and carbohydrates (i) Trypsin treatment. Bacteria were treated with 25 g/ml trypsin in PBS (pH 7.2) at 37°C for 1 h. Cells were washed twice with PBS and resuspended in PBS for testing. (ii) Periodate oxidation. Bacterial cell surface carbohydrates were oxidized by exposure in 0.01 M NaIO4 in 0.05 M sodium acetate, pH 4.2 for 30 min, washed twice with PBS and resuspended in PBS for testing. (iii) Periodate oxidation of TA. A 100 l of TA solution was oxidized by adding 100 l of 0.02 M NaIO4 in 0.10 M sodium acetate pH 4.2. After 30 min, 10 l of ethylene glycol was added, and the pH was adjusted to 7.0 with 1 M Tris–HCl pH 8.0.
Statistics All experiments were performed at least in triplicate and repeated as indicated. Statistical
Teichoic acid and S. epidermidis adhesion
analysis was performed with the Biostatistics program [51] on a PC.
Acknowledgement The authors wish to acknowledge R. A. Proctor for critical reading of this manuscript. This work has been funded by the German Minister for Education and Research (grant 01KI9750/9), by the Medical Faculty of the University of Muenster (grant # HE119840), and by Deutsche Forschungsgemeinschaft, Collaborative Research Center 492, project B9.
References 1 Heilmann C, Hussain M, Peters G, Go¨tz F. Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol 1997; 24: 1013–24. 2 Tojo M, Yamashita DA, Goldman DA, Pier GD. Isolation and characterization of a capsular polysaccharide adhesin from Staphylococcus epidermidis. J Infect Dis 1988; 157: 713–22. 3 Heilmann C, Schweitzer O, Gerke C, Vanittanakom N, Mack D, Go¨tz F. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol Microbiol 1996; 20: 1083–91. 4 Mack D, Fischer W, Krokotsch A, et al. The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear -1,6-linked glukosaminoglykan: Purification and structural analysis. J Bacteriol 1996; 178: 175–83. 5 McKenney D, Hu¨bner J, Muller E, Wang Y, Goldmann DA, Pier GB. The ica locus of Staphylococcus epidermidis encodes production of the capsular polysaccharide/ adhesin. Infect Immun 1998; 66: 4711–20. 6 Hussain M, Herrmann M, von Eiff C, PerdreauRemington F, Peters G. A 140-kilodalton extracellular protein is essential for the accumulation of Staphylococcus epidermidis strains on surfaces. Infect Immun 1997; 65: 519–24. 7 Younger JJ, Christensen GD, Bartley DL, Simmons JCH, Barrett FF. Coagulase-negative staphylococci isolated from cerebrospinal fluid shunts: importance of slime production, species identification, and shunt removal to clinical outcome. J Infect Dis 1987; 156: 548–54. 8 Ziebuhr W, Heilmann C, Go¨tz F, et al. Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. Infect Immun 1997; 65: 890–6. 9 Rupp ME, Archer GL. Coagulase-negative staphylococci: Pathogens associated with medical progress. Clin Infect Dis 1994; 19: 231–45. 10 Refsahl K, Andersen BM. Clinically significant coagulase-negative staphylococci: identification and resistance patterns. J Hosp Infect 1992; 22: 19–31.
269 11 Rupp ME. Coagulase-negative staphylococcal infections: an update regarding recognition and management. Curr Clin Top Infect Dis 1997; 17: 51–87. 12 Whitener C, Caputo GM, Weitekamp MR, Karchmer AW. Endocarditis due to coagulase-negative staphylococci. Microbiologic, epidemiologic, and clinical considerations. Infect Dis Clin North Am 1993; 7: 81–96. 13 Vaudaux PE, Yasuda H, Velazco MI, et al. Role of host and bacterial factors in modulating staphylococcal adhesion to implanted polymer surfaces. J Biomat Appl 1990; 5: 134–53. 14 Flock JI, Gro¨man G, Jo¨nsson K, et al. Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus. EMBO J 1987; 6: 2351–7. 15 Chugh TD, Burns GJ, Shuhaiber HJ, Bahr GM. Adherence of Staphylococcus epidermidis to fibrin platelet clots in vitro mediated by lipoteichoic acid. Infect Immun 1990; 58: 315–9. 16 Aly R, Levit S. Adherence of Staphylococcus aureus to squamous epithelium: role of fibronectin and teichoic acid. Rev Infect Dis 1987; 9 (Suppl 4): S341–50. 17 Hussain M, Peters G, Chhatwal GS, Herrmann M. A lithium chloride-extracted, broad-spectrum-adhesive 42-kilodalton protein of Staphylococcus epidermidis is ornithine carbamoyltransferase. Infect Immun 1999; 67: 6688–90. 18 Hussain M, Wilcox MH, White PJ. The slime of coagulase-negative staphylococci: Biochemistry and relation to adherence. FEMS Microbiol Rev 1993; 104: 191–208. 19 Hussain M, Hastings JG, White JG. Comparison of cellwall teichoic acid with high-molecular-weight extracellular slime material from Staphylococcus epidermidis. J Med Microbiol 1992; 37: 368–75. 20 Green RJ, Davies MC, Roberts CJ, Tendler SJB. Competitive protein adsorption as observed by surface plasmon resonance. Biomaterials 1998; 20: 385–91. 21 Vaudaux PE, Pittet D, Haeberli A, et al. Host factors selectively increase staphylococcal adherence on inserted catheters: a role for fibronectin and fibrinogen/ fibrin. J Infect Dis 1989; 160: 865–75. 22 Vaudaux PE, Francois P, Proctor RA, et al. Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts. Infect Immun 1995; 63: 585–90. 23 Herrmann M, Vaudaux PE, Pittet D, et al. Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material. J Infect Dis 1988; 158: 693–701. 24 Baldassari L, Donelli G, Gelosia A, Simpson AW, Christensen GD. Expression of slime interferes with in vitro detection of host protein receptors of Staphylococcus epidermidis. Infect Immun 1997; 65: 1522–6. 25 Rupp ME, Ulphani JS, Fey PD, Bartscht K, Mack D. Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial-based infection in a mouse foreign body infection model. Infect Immun 1999; 67: 2627–32. 26 Galliani S, Viot M, Cremieux A, Van der Auwera P. Early adhesion of bactermic strains of Staphylococcus epidermidis to polystyrene: influence of hydrophobicity, slime production, plasma, albumin, fibrinogen, and fibronectin. J Lab Clin Med 1994; 123: 685–92.
270 27 Dunne WM, Burd EM. Fibronectin and proteolytic fragments of fibronectin interfere with the adhesion of Staphylococcus epidermidis to plastic. J Appl Bacteriol 1993; 74: 411–6. 28 Muller E, Takeda S, Goldman DA, Pier GB. Blood proteins do not promote adherence of coagulase-negative staphylococci to biomaterials. Infect Immun 1991; 59: 3323–6. 29 Paulsson M, Kober M, Freij LC, Stollenwerk M, Wesslen B, Ljungh A. Adhesion of staphylococci to chemically modified and native polymers, and the influence of preadsorbed fibronectin, vitronectin, and fibrinogen. Biomaterials 1993; 14: 845–53. 30 Brash JL. Hydrophobic polymer surface and their interactions with blood. Ann NY Acad Sci 1977; 283: 356–71. 31 Hasty DL, Courtney HS. Group A streptococcal adhesion. All of the theories are correct. Adv Exp Med Biol 1996; 408: 81–94. 32 Hasty DL, Ofek I, Courtney HS, Doyle RJ. Multiple adhesins of streptococci. Infect Immun 1992; 60: 2147–52. 33 Proctor RA. The staphylococcal fibronectin receptor: Evidence for its importance in invasive infections. Rev Infect Dis 1987; 9: S335–40. 34 Wagner B, Wagner M, Ryc M, Bicova R. Ultrastructural localization of lipoteichoic acid on group A streptococci. In: Kimura Y, Kotani S, Shiokwa S, Eds. Recent advances in streptococci and streptococcal disease. Bracknell: Redbooks Ltd., 1985: 192–4. 35 Ofek I, Simpson WA, Beachey EH. Formation of molecular complexes between structurally defined M protein and acylated and deacylated lipoteichoic acid of Streptococcus pyogenes. J Bacteriol 1982; 149: 426–33. 36 Peters G, Locci R, Pulverer G. Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters. J Infect Dis 1982; 146: 479–82. 37 Vaudaux PE, Waldvogel FA, Morgenthaler JJ, Nydegger UE. Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence. Infect Immun 1984; 45: 768–74. 38 Kessler SW. Use of protein-A bearing staphylococci for the immunoprecipitation and isolation of antigen from cells. Meth Enzymol 1981; 73: 442–58.
M. Hussain et al. 39 Harlow E, Lane D. Antibodies. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1988: 620–1. 40 Naidu AS, Paulsson M, Wadstro¨m T. Particle agglutination assays for rapid detection of fibronectin, fibrinogen, and collagen receptors on Staphylococcus aureus. J Clin Microbiol 1988; 26: 1549–54. 41 Huff E, Oxley H, Silverman CS. Density gradient patterns of Staphylococcus areus cells and cell walls during growth and mechanical disruption. J Bacteriol 1964; 88: 1115–62. 42 Yoshida AC, Heden CG, Cedergren B, Edebo L. A method for the preparation of undigested bacterial cell walls. J Microbiol Technol Eng 1961; 3: 151–9. 43 Archibald AR, Baddiley J, Shaukat GA. The glycerol teichoic acid from walls of Staphylococcus epidermidis 12. Biochem J 1968; 110: 583–8. 44 Hussain M, Hastings JGM, White PJ. Isolation and composition of the extracellular slime made by coagulase-negative staphylococci in a chemically defined medium. J Infect Dis 1991; 163: 534–41. 45 Dubois M, Gilles KA, Hamilton JK, Rabers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956; 28: 350–6. 46 Ames B. Assay of inorganic phosphorus, total phosphorus and phosphates. Meth Enzymol 1966; 8: 115–8. 47 Block RJ, Durrum EL, Zweig G (Eds). A manual of paper chromatography and paper electrophoresis, 2nd edn. New York: Academic Press, 1958: 171–214. 48 Dawson RMC, Elliot DC, Elliot EH, Jones KM (Eds). Methods for detection of biochemical compounds on paper and thin layer chromatograms, with some notes on separation. Data for Biochemical Research. Oxford: Clarendon Press, 1969: 509–91. 49 Carroll JJ, Smith N, Babson AL. A colorimetric serum glucose determination using hexokinase and glucose-6phosphate dehydrogenase. Biochem Med 1971; 4: 171–80. 50 Levvy GA, McAllan A. The N-acetylation and estimation of hexosamines. Biochem J 1959; 73: 127–32. 51 Glantz SA. Biostatistics. New York: McGraw Hill, 2000: 473.
Article available online at http://www.idealibrary.com on
doi:10.1006/mpat.2001.0469
MICROBIAL PATHOGENESIS
Teichoic acid enhances adhesion of Staphylococcus epidermidis to immobilized fibronectin Muzaffar Hussain∗, Christine Heilmann, Georg Peters & Mathias Herrmann† Institute of Medical Microbiology, University Hospital of Muenster, Germany (Received June 11, 2001; accepted in revised form August 30, 2001)
Adhesion is a prerequisite for coagulase-negative staphylococci to cause invasive disease and may be mediated by adhesive host molecules adsorbed on implanted polymers. In this study, we can confirm previous observations demonstrating binding of Staphylococcus epidermidis to fibronectin (FN) adsorbed polymer surfaces. So far, the nature of FN-recognizing adhesin(s) in S. epidermidis remains elusive. Since teichoic acids (TA) have been shown to exert binding functions for extracellular matrix molecules in several Gram-positive species, we have purified wall TA of S. epidermidis laboratory strains KH11 and RP62A, as well as clinical isolate AB9. Using a polymethylmethacrylate (PMMA) coverslip adhesion assay, a microtitre plate assay and a particle agglutination assay, we found that purified TA significantly enhanced adhesion of S. epidermidis KH11 and RP62A to FN coated surfaces. Enhanced adhesion was dose-dependent and saturable. Preincubation, either of microorganisms or of FN coated surfaces, with TA promoted adhesion, while adhesion to TA-adsorbed PMMA was comparably low. This observation may suggest a potential role of cell wall carbohydrates as bridging molecules between microorganisms and immobilized FN in early steps of S. epidermidis pathogenesis. 2001 Academic Press Key words: Staphylococcus epidermidis, bacterial adhesion, fibronectin, teichnoic acid.
Introduction Staphylococcus epidermidis and other coagulasenegative staphylococcal species, previously † Present address: Institute of Medical Microbiology and Hygiene, University of the Saarland Medical School, Bldg. #43, Homburg 66421, Germany. ∗ Author for correspondence. E-mail: muzaffa@uni-muenster. de 0882–4010/01/120261+10 $35.00/0
considered harmless commensals, are now recognized as the most important pathogens for infections of implanted prosthetic material and catheter-associated blood stream infection. The events associated with initiation and course of these infections have been intensely studied, and distinctive mechanisms contributing to adhesion of microorganisms to the uncoated polymer [1, 2], proliferation and accumulation on the surface resulting in biofilm formation [3–6], and persistence of the sessile bacterial population until 2001 Academic Press
262 0.7
(a)
Adhesion (%)
0.6 0.5 0.4 0.3 0.2 0.1 0.0
1.0
KH11
RP62A
AD22
AB7
AB9
RP62A
AD22
AB7
AB9
(b)
0.9 0.8 0.7 OD460 nm
removal of the polymer material [7, 8] have been identified. Generally, it is thought that S. epidermidis prosthetic device infections occur through colonization of naked polymer surfaces by microorganisms resident in the surgical or the catheter wound [9]. However, in addition to indwelling device infection, S. epidermidis are also capable of causing invasive disease, such as osteomyelitis [10], deep tissue infection [11] or native valve endocarditis [12]. Therefore, in these entities as well as in certain types of polymer-associated infection (e.g., tunnel infections of implanted catheters or ventricular assist device drive lines), S. epidermidis may bind to immobilized host factors rather than to unadsorbed plastic [13]. In vitro studies have demonstrated that S. epidermidis adheres to polymeradsorbed fibronectin (FN), yet, in contrast to the FN-binding proteins in S. aureus [14], a FNbinding adhesin in S. epidermidis has not been identified. Teichoic acid (TA), an essential wall constituent of staphylococci, has been implicated in the binding of S. epidermidis binding fibrin clots [15] and in the adhesion of S. aureus to epithelial cells [16]. Therefore, the goal of this study was to evaluate a potential role of S. epidermidis TA as a candidate molecule mediating binding of S. epidermidis to immobilized FN.
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0.6 0.5 0.4 0.3 0.2 0.1 0.0
KH11
Figure 1. Promotion of adhesion of five strains of S. epidermidis to FN-adsorbed surfaces. (a) PMMA coverslips (b) microtitre plate wells. Binding to BSA (Φ) used as control and to FN (Ε). Experimental conditions described in Materials and Methods.
Results Interaction of S. epidermidis with soluble and immobilized FN (i) Coagglutination test. S. epidermidis bound soluble FN as demonstrated in a coagglutination reaction using formalin fixed and heat killed Staphylococcus aureus Cowan 1 cells sensitized with anti-FN-Abs. All five tested S. epidermidis isolates revealed a positive agglutination reaction, yet agglutination occurred to a different extent with strains KH11 and AD22 yielding a (+++), strains RP62A and AB7 a (++) and strain AB9 a (+) reaction. (ii) Particle agglutination test. Using FN coated latex particles results similar to those of coagglutination test results were obtained. Two strains, KH11 and AD22, showed strong agglutination reaction (+++), two strains, RP62A and AB7, agglutinated moderately (++) and a fifth strain, AB9, agglutinated weakly (+). (iii) Adhesion to
FN coated PMMA cover slips. Pre-adsorption of FN to PMMA promoted adhesion of the five S. epidermidis isolates tested in this study [Fig. 1(a)]. (iv) Adhesion to FN coated microtitre plate. Promotion in adhesion of these isolates to FNadsorbed surfaces was also observed when using a polystyrene microtitre plate adhesion assay [Fig. 1(b)]. Adhesion of strain KH11 to FN coated polystyrene surfaces was found to be dosedependent with concentrations of FN 5–50 g/ ml (data not shown).
Effect of treatment of bacteria with trypsin and periodate oxidation on adhesion This experiment was performed to characterize the nature of the receptor for FN on staphylococcal cells surface. In separate experiments bacterial cell surface proteins were removed by
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Table 1. Adhesion of S. epidermidis to FN-adsorbed PMMA or FN-adsorbed polystyrene and effect of soluble TA Radiometric adhesion assay
KH11 RP62A AD22 AB7 AB9
Microtitre plate assay
Albumin FN FN plus TA Percent adhesion (mean, SD)
Albumin
0.03 0.09 0.04 0.06 0.09
0.09 0.08 0.04 0.08 0.05
(0.01) (0.07) (0.02) (0.05) (0.06)
0.51 0.57 0.65 0.27 0.39
(0.05) (0.06) (0.02) (0.03) (0.01)
1.74 1.74 1.59 0.74 0.45
(0.11) (0.12) (0.16) (0.10) (0.08)
trypsination and carbohydrates were removed by periodate oxidation. The adhesion reaction was found to be both trypsin and periodatesensitive, because pre-treatment of bacteria with these reagents caused a reduction of KH11 adhesion to FN-PMMA by 65 and 74%, and of RP62A by 61 and 63%, respectively. The effect of trypsin suggested a possible role of bacterial surface proteins in the interaction with immobilized FN, adhesion assays were performed using a mutant defective in expression of AtlE. AtlE is an autolysin of strain S. epidermidis O47 with vitronectin (VN) binding activity in Western-ligand assays [1], and the AtlE analogue from strain S. epidermidis strain AB9 recognized both FN and VN [17]. Adhesion of the mutant to FN-PMMA was slightly elevated (mean adhesion, 0.86%) compared to the wild-type strain (0.34%), but this was accompanied by slightly enhanced adhesion to BSA-PMMA (0.11 vs 0.06%). Thus, AtlE does not appear to mediate adhesion of S. epidermidis O-47 to FN-adsorbed surfaces.
(0.02) (0.50) (0.03) (0.03) (0.04)
FN FN plus TA OD492nm (mean, SD) 0.22 0.53 0.57 0.10 0.32
(0.06) (0.04) (0.13) (0.01) (0.04)
0.54 0.79 0.93 0.42 0.56
(0.08) (0.09) (0.04) (0.03) (0.10)
p-anisidine or AgNO3/NaOH. In another chromatogram, a glycerol–phosphate spot was disclosed with ammonium molybdate+perchloric acid+HCl mixture. The chromatogram stained with ninhydrin showed only one spot close to the alanine marker. Hydrolysates prepared from isolated TA from S. epidermidis RP62A, KH11 and AB9 did exhibit these characteristics typical for staphylococcal wall TA [18, 19]. (ii) Enzymatic and chemical analysis. In TA from strain RP62A, 165 g of glucose and 40 g of glucosamine were determined per mg of freezedried material. Taking molecular masses of polymer units of glucose and glucosamine as 162 and 203, respectively, then these data suggests presence of 1 M of glucose and 0.2 M of glucosamine in each mg of freeze-dried material. (iii) Reaction with lectins. TA isolated from three strains (RP62A, KH11 and AB9) showed strong precipitation reactions with lectins concanavalin-A and lectin from Triticum vulgaris (wheat germ lectin) specific for glucose and glucosamine respectively. The reaction with lectins suggests presence of glucose and glucosamine as glucosy substituents.
Purification and analysis of TA (i) Thin layer chromatography. Cell walls isolated from the middle layer of the sucrose gradient were hydrolysed in 6 M HCl for 18 h at 105°C, and amino acids were separated by thin layer chromatography. Only alanine, glycine, glutamic acid and lysine were detected with ninhydrin, a clear indication of a pure cell wall preparation. TA extracted from purified cell walls by either procedure was hydrolysed in 3 M HCl for 3 h and analysed by thin layer chromatography. Spots having Rglucose values close to those of glucose, glucosamine and glycerol–phosphate were identified with
Role of TA in the FN–S. epidermidis interaction These findings prompted further evaluation of the role of cell wall TA to the binding reaction. Upon addition of TA purified by anion exchange chromatography (final concentration, 16.5 g/ ml), an increase in adhesion of all five tested isolates, both in the radiometric adhesion assay as well as in the microtitre plate assay, were observed with strain AB9 being least promoted (Table 1). Using the particle agglutination assay, addition of TA purified from either RP62A, AB9
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M. Hussain et al. (a) 2.0
***
3.0 (b) 2.5
***
2.0 Adhesion (%)
Adhesion (%)
1.5
1.0
1.5 1.0
0.5 0.5
0.0
KH11
RP62A
0.0
KH11
Figure 2. (a) Effect of different TA preparations on promotion of S. epidermidis adhesion to FN-adsorbed PMMA. Untreated (Φ) or FN-pre-adsorbed (25 g in 1 ml PBS, 60 min, 37°C) (Ε Φ, Ε, and Ε Φ) PMMA coverslips were incubated with 4×106 cfu/ml of [3H]-thymidine-labelled bacteria in 1 ml of Ca++/Mg++PBS supplemented with 0.5% human serum albumin (60 min, 37°C, shaking water bath), then washed. The number of adherent microorganisms was expressed as percentage of inoculum. In some experiments, suspensions containing radiolabelled bacteria and FN-PMMA were supplemented with TA purified from the DEAE cellulose column (Ε) or from the concanavalin-A column (Ε Φ) (16.5 g/ml, respectively) prepared as described in Materials and Methods. Shown are means of three determinations±SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) and subsequent Student-Newman-Keuls test for determination of the significance levels of differences between observations. ANOVA, P<0.001; ∗∗∗, P<0.01 of FN alone vs DEAE- or concavalin-A-eluate supplement. (b) Effect of two TA preparations on adhesion of S. epidermidis KH11 to FN-PMMA. TA was prepared either from S. epidermidis KH11 (Ε) or from RP62A (Ε Φ) and used to supplement the adhesion buffer as described in Fig. 1.
or KH11 to FN coated beads resulted in a moderate agglutination (+/++), yet, when FNbeads were first treated with purified TA and then incubated with washed cells of strains KH11, RP62A or AB9, an immediate and strong (+++) agglutination reaction was detected. Control (BSA-adsorbed) beads revealed no agglutination whether incubated with TA alone or with TA plus bacterial cells. Strains KH11 and RP62A were further studied. Lectin-affinity chromatography did not enhance the biologic activity of purified TA, since TA-enhanced adhesion was similar using either purification procedure [Fig. 2(a)]. No discernible difference in adhesion was observed when TA isolated upon either TA extraction procedure (trichloroacetic acid or lysostaphin–lysozyme extraction) was used (not shown). For subsequent experiments, TA was prepared from digestion of cell walls with lysostaphin–lysozyme and purified by anion-exchange chromatography. The promotion of adhesion by TA was not isolate-dependent, i.e. TA purified from strain RP62A promoted adhesion of strain KH11 to a similar
extent when compared with TA from strain KH11 [Fig. 2(b)]. Adhesion was found to be dosedependent with concentrations of >16.5 g/ml, resulting in saturation of TA-mediated adhesion enhancement (Fig. 3). While these experiments demonstrated the adhesion-promoting effect of TA in co-incubation with FN-PMMA and staphylococci (Fig. 4), this effect was also to be observed, albeit to a lesser extent, if PMMA coverslips were preincubated with TA then rinsed and tested for adhesion. Moreover, the extent of adhesion of bacteria pre-adsorbed with TA then washed was found to be almost identical to adhesion to FN-PMMA in the presence of TA. In contrast, microorganisms adhered to PMMA pre-adsorbed with TA only to a small extent.
Interaction of TA with DIG-labelled ligands DIG labelled FN bound very well to TA and DIG labelled FG bound to a lesser extent to immobilized TA on a nitrocellulose membrane, while DIG labelled VN did not show any binding to TA (data not shown).
Teichoic acid and S. epidermidis adhesion
265
addition of oxidized TA are: strain KH11 OD492nm, 0.24±0.05 (mean±SD, n=3), 0.25 (0.08); strain AB9 0.36 (0.07), 0.35 (0.05).
1.4
Adhesion (%)
1.2 1.0 0.8
Effect of soluble FN on adhesion to TA coated polystyrene surface
0.6 0.4 0.2 1.65
16.5 Teichoic acid added (µg/ml)
165
Figure 3. Adhesion promotion by TA as a function of TA concentration. Various concentrations of TA were supplemented to the adhesion buffer as described in Fig. 1, and adhesion of radiolabelled S. epidermidis KH11 was subsequently determined. Shown are means of triplicate determinations±SD.
***
When a bacterial suspension containing 50 g/ ml FN was added to a microtitre plate wells precoated with TA, a slightly enhanced adhesion of strain KH11 and AB9 was observed: strain KH11, OD492nm, 0.09±0.03 (mean±SD, n=3) enhanced to 0.14 (0.05) and strain AB9, 0.07 (0.04) enhanced to 0.12 (0.05). The possible explanation includes: (i) a small amount of immobilized TA on a polystyrene surface or (ii) the fact that functional binding sites of TA may not be any more available for bridging between cell surface components and soluble FN after immobilization of TA.
***
Adhesion (%)
2.0
Discussion
1.5
1.0
0.5
0.0
KH11
RP62A
Figure 4. Effect of coincubation vs preincubation of TA either with FN-PMMA or with bacteria. Control assays either without FN (Φ) or with FN-PMMA alone (Ε Φ) were performed as described in Fig. 1. Alternatively, TA (16.5 g/ml) was either added during the adhesion incubation (Ε), or either FN-PMMA (Ε Φ) or radiolabelled bacteria (P) were preincubated with TA, then washed and incubated for bacterial adhesion. In some experiments, PMMA coverslips without FN were incubated in the presence of TA (16.5 g/ml) and radiolabelled bacteria (Q). Shown are means±SD of three determinations. ANOVA, P<0.001, ∗∗∗, P<0.01 of FN-PMMA alone vs coincubation or preincubation with TA.
Effect of oxidized TA on adhesion The oxidized TA failed to enhance adhesion of strains KH11 and AB9 to polystyrene microtitre plate coated with FN. Values before and after
In this study, we describe the interaction of S. epidermidis with FN and identify cell wall TA as an adhesive molecule recognizing adsorbed FN. After contact with blood, plasma proteins are sequentially adsorbed to polymeric surfaces, with high-abundant, low molecular-weight proteins, such as albumin, being rapidly and preferentially adsorbed but subsequently replaced by less abundant, more surface active proteins such as FN and fibrinogen [20]. These observations are in good agreement with data obtained from ex vivo human [21] and canine [22] catheter materials witnessing deposition of significant amounts of adhesive molecules on the polymer substrates. Accordingly, various studies on the role of FN on S. epidermidis adhesion, albeit with contrasting results, have been reported [21, 23–29], while adhesion to other immobilized matrix proteins was found to show more interstrain variability or to be low [23]. Experimental conditions contributing to differences in physicochemical binding forces, notably in surface hydrophobicity, may account at least for a part of these differences [1, 26, 30]. To model the physiologic conditions addressed in this study, we have performed our assays in a physiologic, i.e. albumin-containing milieu, and by using three different types of assays we
266
have confirmed that polymer-adsorbed FN does indeed promote adhesion of S. epidermidis to the substrate. Recently, we have reported on putative S. epidermidis adhesins recognizing FN in Westernligand blots [17]. While one of the released molecules was ornithine carbamoyltransferase, an intracellular enzyme not involved in adhesion to surfaces, here we present evidence that another S. epidermidis protein recognizing VN and FN in Western-ligand assays, the S. epidermidis autolysin AtlE, does not contribute to adhesion to FN. Thus, a proteinaceous adhesin recognizing FN remains unidentified in S. epidermidis. Gram-positive cocci have been shown to interact with whole cells or extracellular matrix protein, notably FN, via TA and/or lipoteichoic acid. In numerous studies {reviewed in [31]}, streptococcal lipoteichoic acid (LTA) has been suggested to bind to FN and these observations have prompted a model of streptococcal attachment to extracellular matrix or eukaryotic cells involving a two-step mechanism including LTA and proteinaceous receptors [32]. This model has been challenged but provides a basis for further research in multiple-step adhesion mechanisms of Gram-positive cocci including staphylococci [15, 16, 33]. In general, research on the role of cell wall TA as adhesins is hampered by the fact that mutants of staphylococci deficient in TA are not available, since wall TA appear to be essential for survival. Our evidence suggesting a role of TA as an S. epidermidis adhesin for FN is based on the following observations: (i) the demonstration of dose-dependency and saturability of TAenhanced adhesion of S. epidermidis to adsorbed FN, (ii) the fact that S. epidermidis adheres only slightly to TA-pre-adsorbed PMMA in the absence of FN, and (iii) the effect of carbohydratemodifying periodate on adhesion. As protease treatment also reduced adhesion, both proteinand carbohydrate-type ligands may be involved, however, it has been demonstrated for streptococci that trypsin digestion of bacteria removes most of the TA from cell surface [34]. Our findings suggest that TA forms a bridge between the FN molecule and the S. epidermidis cell surface. This may occur via rebinding of TA through its anionic backbone to cationic molecules on cell surface [35], however, additional research, e.g. using mutants defective in TA-modifying enzymes, is necessary to further determine the molecular mechanisms involved. Further work
M. Hussain et al.
is also necessary to explore the potential of these findings for novel prophylactic or therapeutic strategies directed against S. epidermidis infections.
Materials and Methods Bacterial strains and culture media S. epidermidis isolates KH11 [36] and RP62A (ATCC 35984) were used. In addition, clinical S. epidermidis blood stream isolates AD22, AB7 and AB9 were selected from the strain collection of our institute for further study. For determination of the role of AtlE on adhesion to FN, in some experiments wild-type strain O-47 and its AtlEdeficient mutant, mut1 [1], was also used. Identification of clinical isolates as S. epidermidis on a species level was performed using the ID 32 biochemical test kit (BioMerieux, Marcy-l’Etoile, France). Working cultures were maintained on blood agar plates (for strain mut1 supplemented with erythromycin 10 g/ml), and fresh overnight cultures were grown on brain–heart broth/ agar (Merck, Darmstadt, Germany) and Muller Hinton broth/agar (Mast, Merseyside, U.K.).
Solid phase adhesion assay For radiomateric analysis of S. epidermidis adhesion to coated solid surfaces, a previously described assay was used [37]. Briefly, solution containing 25 g/ml human plasma FN (Chemicon, Temecula, CA, U.S.A.) was allowed to adsorb to PMMA coverslips for 60 min at 37°C. Thereafter, coverslips were washed with PBS and incubated in a shaking water bath with [3H] thymidine-labelled S. epidermidis cells [4×106 cfu in 1 ml PBS containing 0.5% bovine serum albumin (Sigma-Aldrich, Deisenhofen, Germany)] for 60 min at 37°C. After adhesion, the PMMA coverslips were washed with PBS three times, and adherent cpm counts were determined. In some experiments TA was added as g of glucose per ml of adhesion assay.
Microtitre plate adhesion assay Polystyrene plates (96 well) (Greiner, Frickenhausen, Germany) were coated with FN (50 g/ml−1 in 50 mM sodium carbonate buffer,
Teichoic acid and S. epidermidis adhesion
pH 9.6) for 18 h at 4°C, then blocked with 3% BSA in TBS (25 mM Tris–HCl, 100 mM NaCl, pH 7.5) and washed. Bacteria were grown for 18 h at 37°C, washed once with TBS, adjusted to an OD578nm 1.0 in TBS, and to each well 200 l of bacterial suspension was added. Plates were incubated at 37°C for 1 h, then wells were washed three times, stained with safranin for 1 min, and the plate was read at 492 nm on a Titertek-multiscan-8 (Flow laboratories, Bonn, Germany). To study the effect of soluble FN on adhesion to TA coated polystyrene surface, the above detailed experiment was performed with the following two modifications: (i) the surface was coated with TA (16.5 g/ml) and (ii) the bacterial suspension contained 50 g/ml FN.
Coagglutination test The preparation of coagglutination reagent (SAC+anti-FN-Abs) involved fixing of protein A found on surface of S. aureus cowan1 (ATCC 12598) cells with formaldehyde and then killing bacteria by heating [38, 39]. To the resulting protein A solid phase, anti-FN-Abs (Dako) were coupled. Bacteria were grown in BHI for 18 h at 37°C with shaking, washed twice with PBS and incubated at room temperature after suspension in PBS containing 50 g/ml FN. After 1 h, FN treated bacteria were washed twice with PBS and resuspended in PBS to OD598nm of 1.0. For the coagglutination test, 10 l of FN exposed bacterial suspension was mixed with 20 l of coagglutination reagent on a microscope slide. After 3 min reactions were scored as negative (−) or from weakly positive (+) to strongly positive (+++). No agglutination was found when the PBS used to suspend bacteria and coagglutination reagent was tested as a control.
Particle agglutination assay Method of Naidu et al. [40] was used. Briefly, 200 l of blue latex beads (Sigma) were preadsorbed with FN (100 g/ml) or with BSA for control in sodium carbonate buffer (pH 9.5), then washed. Bacteria were grown overnight in BHI, washed, and resuspended in PBS. FN-latex beads (20 l) were mixed with 20 l of bacterial suspension, and after 3 min the agglutination reaction was scored as negative (−), or from weak positive (+) to strong positive (+++).
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Preparation of cell walls For isolation of cell wall, bacteria were grown in 800 ml TSB in two 1 l flasks, each inoculated with 0.5 ml of an 18 h culture in the same medium and incubated with shaking at 37°C for 18 h. Bacteria were pelleted by centrifugation (10 000×g, 10 min, 4°C) and washed twice with distilled water. Washed bacteria were broken by the method of Huff et al. [41], shaking with glass beads in a Braun cell homogenizer (Braun, Melsungen, Germany). For the separation of walls by sucrose gradient centrifugation, the method of Yoshida et al. [42] was used. Cell walls from the middle layer of sucrose gradient were resuspended in 50 mM Tris–HCl pH 7.8 plus trypsin 250 g/ml, and kept at 37°C with shaking for 4 h. The suspension was centrifuged (32 000×g, 30 min, 4°C), sedimented walls were washed with 1 M NaCl, then six times with distilled water, and freeze-dried. For characterization, freeze-dried cell walls (5 mg) in 6 M HCl (1 ml) in a sealed glass tube were heated at 105°C in an oven for 18 h. Acid hydrolysate was dried in a dessicator over silica gel in presence of NaOH under vacuum. Acid from hydrolysate was completely removed by suspending the dried hydrolysate in a few drops of water and repeating the drying step at least three times. Amino acids were separated by thin layer chromatography and detected by ninhydrin spray.
Isolation of TA from cell walls TA was extracted from cell walls by two methods: (i) digestion with lysozyme+ lysostaphin. Cell walls (1 mg/ml) were suspended in 50 mM Tris–HCl, pH 8, containing 1.2 mM EDTA (Sigma) and 0.145 M NaCl (Merck, Darmstadt, Germany). The suspension was first digested with a mixture of recombinant lysostaphin (Applied Micro Inc., New York, U.S.A.) and lysozyme (Merck) for 3 h at 37°C, followed by 1 mg/ml trypsin (Sigma) digestion for 4 h at 37°C. After enzyme treatment, the suspension was centrifuged (30 000×g, 4°C, 30 min), then passed through a 0.22 m membrane filter (Millipore Corporation, Bedford, U.S.A.) to remove cell wall fragments. Five volume of ethanol was added to the filtrate. After overnight equilibration at 4°C, the liquid was discarded and the precipitate containing TA was freeze-dried. (ii) Extraction with trichloroacetic
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acid (TCA) by the method of Archibald et al. [43].
Anion-exchange and affinity chromatography TA was fractionated by ion-exchange chromatography on DEAE-cellulose and affinity chromatography as described previously for the fractionation of extracellular products [19, 44]. (i) Briefly, the DEAE cellulose (Sigma) column (1.5×11 cm) was equilibrated with 0.1 M ammonium acetate (Merck) buffer (pH 5.5), and 25 mg of crude TA in 2 ml buffer was loaded onto the column. The column was eluted stepwise with increasing concentrations of NaCl in buffer. Fractions were analysed by a phenol/ sulfuric acid assay [45] and an assay for total phosphorus [46]. Fractions making peaks were pooled, dialysed against distilled water for 24 h, and freeze-dried. (ii) Affinity chromatography. Material eluted with 0.5 M NaCl in 50 mM ammonium acetate from DEAE-cellulose was loaded on a concanavalin A sepharose 4-B column (Sigma) equilibrated with 0.1 M sodium acetate (pH 6.0) containing 1 M NaCl, 1 mM of each CaCl2, MgCl2, MnCl2 and 0.02% NaN3. The column was washed with 20 bed volumes of loading buffer, then bound material was eluted with five bed volumes of 0.1 M methyl -Dglucopyranoside in loading buffer, and assayed for phosphorus. The eluted material was dialysed against distilled water then freeze-dried.
Analysis of TA by thin layer chromatography TA was hydrolysed in 3 M HCl for 3 h at 100°C in a boiling water bath. Acid from hydrolysate was removed as detailed earlier for cell wall hydrolysis. Prepared hydrolysate was separated on thin layer chromatography plates and detected using standard methods [47, 48].
Estimation of glucose and glucosamine in TA preparation Glucose concentration was determined by a highly specific hexokinase method of Carroll et al. [49] using glucose kit 115-A (Sigma). For measurement of glucosamine, method of Levvy
M. Hussain et al.
and McAllan [50] with glucosamine. HCl (Sigma) as standard was used.
Reaction with lectins Two lectins, concanavalin-A (Sigma) and lectin from Triticum vulgaris (wheat germ lectin; Sigma), were used. TA suspension (20 l) was added to lectin solution (10 mg/ml, 20 l) on a glass slide. Results were recorded as negative or positive (clumping) after 1 min.
Interaction of TA with FN TA was immobilized on nitrocellulose membrane (Schleicher & Scuell, Dasel, Germany) by allowing a 5 l TA solution to penetrate the membrane and subsequently dried. The remaining binding sites on the membrane were blocked by 3% BSA (fraction V, Sigma), then separate membranes were probed with DIGlabelled FN, FG or VN for 1 h. The membranes were washed three times with TBS-T and transferred to a solution of alkaline phosphataseconjugated anti-DIG-Abs (Roche, Mannheim, Germany) for 1 h. Spots binding FN, FG or VN were detected in an alkaline phosphatase colour reaction (Bio-Rad) in accordance with the instructions of the supplier.
Removal of surface proteins and carbohydrates (i) Trypsin treatment. Bacteria were treated with 25 g/ml trypsin in PBS (pH 7.2) at 37°C for 1 h. Cells were washed twice with PBS and resuspended in PBS for testing. (ii) Periodate oxidation. Bacterial cell surface carbohydrates were oxidized by exposure in 0.01 M NaIO4 in 0.05 M sodium acetate, pH 4.2 for 30 min, washed twice with PBS and resuspended in PBS for testing. (iii) Periodate oxidation of TA. A 100 l of TA solution was oxidized by adding 100 l of 0.02 M NaIO4 in 0.10 M sodium acetate pH 4.2. After 30 min, 10 l of ethylene glycol was added, and the pH was adjusted to 7.0 with 1 M Tris–HCl pH 8.0.
Statistics All experiments were performed at least in triplicate and repeated as indicated. Statistical
Teichoic acid and S. epidermidis adhesion
analysis was performed with the Biostatistics program [51] on a PC.
Acknowledgement The authors wish to acknowledge R. A. Proctor for critical reading of this manuscript. This work has been funded by the German Minister for Education and Research (grant 01KI9750/9), by the Medical Faculty of the University of Muenster (grant # HE119840), and by Deutsche Forschungsgemeinschaft, Collaborative Research Center 492, project B9.
References 1 Heilmann C, Hussain M, Peters G, Go¨tz F. Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol 1997; 24: 1013–24. 2 Tojo M, Yamashita DA, Goldman DA, Pier GD. Isolation and characterization of a capsular polysaccharide adhesin from Staphylococcus epidermidis. J Infect Dis 1988; 157: 713–22. 3 Heilmann C, Schweitzer O, Gerke C, Vanittanakom N, Mack D, Go¨tz F. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol Microbiol 1996; 20: 1083–91. 4 Mack D, Fischer W, Krokotsch A, et al. The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear -1,6-linked glukosaminoglykan: Purification and structural analysis. J Bacteriol 1996; 178: 175–83. 5 McKenney D, Hu¨bner J, Muller E, Wang Y, Goldmann DA, Pier GB. The ica locus of Staphylococcus epidermidis encodes production of the capsular polysaccharide/ adhesin. Infect Immun 1998; 66: 4711–20. 6 Hussain M, Herrmann M, von Eiff C, PerdreauRemington F, Peters G. A 140-kilodalton extracellular protein is essential for the accumulation of Staphylococcus epidermidis strains on surfaces. Infect Immun 1997; 65: 519–24. 7 Younger JJ, Christensen GD, Bartley DL, Simmons JCH, Barrett FF. Coagulase-negative staphylococci isolated from cerebrospinal fluid shunts: importance of slime production, species identification, and shunt removal to clinical outcome. J Infect Dis 1987; 156: 548–54. 8 Ziebuhr W, Heilmann C, Go¨tz F, et al. Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. Infect Immun 1997; 65: 890–6. 9 Rupp ME, Archer GL. Coagulase-negative staphylococci: Pathogens associated with medical progress. Clin Infect Dis 1994; 19: 231–45. 10 Refsahl K, Andersen BM. Clinically significant coagulase-negative staphylococci: identification and resistance patterns. J Hosp Infect 1992; 22: 19–31.
269 11 Rupp ME. Coagulase-negative staphylococcal infections: an update regarding recognition and management. Curr Clin Top Infect Dis 1997; 17: 51–87. 12 Whitener C, Caputo GM, Weitekamp MR, Karchmer AW. Endocarditis due to coagulase-negative staphylococci. Microbiologic, epidemiologic, and clinical considerations. Infect Dis Clin North Am 1993; 7: 81–96. 13 Vaudaux PE, Yasuda H, Velazco MI, et al. Role of host and bacterial factors in modulating staphylococcal adhesion to implanted polymer surfaces. J Biomat Appl 1990; 5: 134–53. 14 Flock JI, Gro¨man G, Jo¨nsson K, et al. Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus. EMBO J 1987; 6: 2351–7. 15 Chugh TD, Burns GJ, Shuhaiber HJ, Bahr GM. Adherence of Staphylococcus epidermidis to fibrin platelet clots in vitro mediated by lipoteichoic acid. Infect Immun 1990; 58: 315–9. 16 Aly R, Levit S. Adherence of Staphylococcus aureus to squamous epithelium: role of fibronectin and teichoic acid. Rev Infect Dis 1987; 9 (Suppl 4): S341–50. 17 Hussain M, Peters G, Chhatwal GS, Herrmann M. A lithium chloride-extracted, broad-spectrum-adhesive 42-kilodalton protein of Staphylococcus epidermidis is ornithine carbamoyltransferase. Infect Immun 1999; 67: 6688–90. 18 Hussain M, Wilcox MH, White PJ. The slime of coagulase-negative staphylococci: Biochemistry and relation to adherence. FEMS Microbiol Rev 1993; 104: 191–208. 19 Hussain M, Hastings JG, White JG. Comparison of cellwall teichoic acid with high-molecular-weight extracellular slime material from Staphylococcus epidermidis. J Med Microbiol 1992; 37: 368–75. 20 Green RJ, Davies MC, Roberts CJ, Tendler SJB. Competitive protein adsorption as observed by surface plasmon resonance. Biomaterials 1998; 20: 385–91. 21 Vaudaux PE, Pittet D, Haeberli A, et al. Host factors selectively increase staphylococcal adherence on inserted catheters: a role for fibronectin and fibrinogen/ fibrin. J Infect Dis 1989; 160: 865–75. 22 Vaudaux PE, Francois P, Proctor RA, et al. Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts. Infect Immun 1995; 63: 585–90. 23 Herrmann M, Vaudaux PE, Pittet D, et al. Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material. J Infect Dis 1988; 158: 693–701. 24 Baldassari L, Donelli G, Gelosia A, Simpson AW, Christensen GD. Expression of slime interferes with in vitro detection of host protein receptors of Staphylococcus epidermidis. Infect Immun 1997; 65: 1522–6. 25 Rupp ME, Ulphani JS, Fey PD, Bartscht K, Mack D. Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial-based infection in a mouse foreign body infection model. Infect Immun 1999; 67: 2627–32. 26 Galliani S, Viot M, Cremieux A, Van der Auwera P. Early adhesion of bactermic strains of Staphylococcus epidermidis to polystyrene: influence of hydrophobicity, slime production, plasma, albumin, fibrinogen, and fibronectin. J Lab Clin Med 1994; 123: 685–92.
270 27 Dunne WM, Burd EM. Fibronectin and proteolytic fragments of fibronectin interfere with the adhesion of Staphylococcus epidermidis to plastic. J Appl Bacteriol 1993; 74: 411–6. 28 Muller E, Takeda S, Goldman DA, Pier GB. Blood proteins do not promote adherence of coagulase-negative staphylococci to biomaterials. Infect Immun 1991; 59: 3323–6. 29 Paulsson M, Kober M, Freij LC, Stollenwerk M, Wesslen B, Ljungh A. Adhesion of staphylococci to chemically modified and native polymers, and the influence of preadsorbed fibronectin, vitronectin, and fibrinogen. Biomaterials 1993; 14: 845–53. 30 Brash JL. Hydrophobic polymer surface and their interactions with blood. Ann NY Acad Sci 1977; 283: 356–71. 31 Hasty DL, Courtney HS. Group A streptococcal adhesion. All of the theories are correct. Adv Exp Med Biol 1996; 408: 81–94. 32 Hasty DL, Ofek I, Courtney HS, Doyle RJ. Multiple adhesins of streptococci. Infect Immun 1992; 60: 2147–52. 33 Proctor RA. The staphylococcal fibronectin receptor: Evidence for its importance in invasive infections. Rev Infect Dis 1987; 9: S335–40. 34 Wagner B, Wagner M, Ryc M, Bicova R. Ultrastructural localization of lipoteichoic acid on group A streptococci. In: Kimura Y, Kotani S, Shiokwa S, Eds. Recent advances in streptococci and streptococcal disease. Bracknell: Redbooks Ltd., 1985: 192–4. 35 Ofek I, Simpson WA, Beachey EH. Formation of molecular complexes between structurally defined M protein and acylated and deacylated lipoteichoic acid of Streptococcus pyogenes. J Bacteriol 1982; 149: 426–33. 36 Peters G, Locci R, Pulverer G. Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters. J Infect Dis 1982; 146: 479–82. 37 Vaudaux PE, Waldvogel FA, Morgenthaler JJ, Nydegger UE. Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence. Infect Immun 1984; 45: 768–74. 38 Kessler SW. Use of protein-A bearing staphylococci for the immunoprecipitation and isolation of antigen from cells. Meth Enzymol 1981; 73: 442–58.
M. Hussain et al. 39 Harlow E, Lane D. Antibodies. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1988: 620–1. 40 Naidu AS, Paulsson M, Wadstro¨m T. Particle agglutination assays for rapid detection of fibronectin, fibrinogen, and collagen receptors on Staphylococcus aureus. J Clin Microbiol 1988; 26: 1549–54. 41 Huff E, Oxley H, Silverman CS. Density gradient patterns of Staphylococcus areus cells and cell walls during growth and mechanical disruption. J Bacteriol 1964; 88: 1115–62. 42 Yoshida AC, Heden CG, Cedergren B, Edebo L. A method for the preparation of undigested bacterial cell walls. J Microbiol Technol Eng 1961; 3: 151–9. 43 Archibald AR, Baddiley J, Shaukat GA. The glycerol teichoic acid from walls of Staphylococcus epidermidis 12. Biochem J 1968; 110: 583–8. 44 Hussain M, Hastings JGM, White PJ. Isolation and composition of the extracellular slime made by coagulase-negative staphylococci in a chemically defined medium. J Infect Dis 1991; 163: 534–41. 45 Dubois M, Gilles KA, Hamilton JK, Rabers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956; 28: 350–6. 46 Ames B. Assay of inorganic phosphorus, total phosphorus and phosphates. Meth Enzymol 1966; 8: 115–8. 47 Block RJ, Durrum EL, Zweig G (Eds). A manual of paper chromatography and paper electrophoresis, 2nd edn. New York: Academic Press, 1958: 171–214. 48 Dawson RMC, Elliot DC, Elliot EH, Jones KM (Eds). Methods for detection of biochemical compounds on paper and thin layer chromatograms, with some notes on separation. Data for Biochemical Research. Oxford: Clarendon Press, 1969: 509–91. 49 Carroll JJ, Smith N, Babson AL. A colorimetric serum glucose determination using hexokinase and glucose-6phosphate dehydrogenase. Biochem Med 1971; 4: 171–80. 50 Levvy GA, McAllan A. The N-acetylation and estimation of hexosamines. Biochem J 1959; 73: 127–32. 51 Glantz SA. Biostatistics. New York: McGraw Hill, 2000: 473.