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New concepts in the pathogenesis of staphylococcal central venous catheter infections
NEW CONCEPTS IN THE PA THOGENESIS :)F S T A P H Y L O C O C C A L CENTRAL VENOUS ',.4T H E T E R I N F E C T I O N S -IERRMANN, G. PETERS
•
Introduction
Central venous catheters are indispensable in modern intensive care, since they are used for a large variety of purposes, including administration of fluids and electrolytes, blood products, drugs, for total parenteral nutrition, and for hemodynamic monitoring. Polyurethanes and polyvinylchloride are among the most frequently used materials for these devices. However, microbial colonization of intravenous lines is the single most important source for nosocomial bacteremia with its potential consequences of sepsis and metastic infection. It contributes significantly to hospital-associated morbidity and mortality. Although bacteria such as Pseudomonas, other nonfermenting gram-negative rods, enterobacteriaceae, diphtheroids and enterococci as well as fungi are causing intravenous catheter infection, staphylococci, particularly coagulase-negative staphylococci (CONS) but also S. aureus, are by far the most important since most frequently isolated strains [1]. In the past, interactions between material and bacteria have been most extensively studied for staphylococci, and although many of the pathogenic concepts in catheter infection may also be valid for other bacterial species, this contribution will focus on the pathogenic events occuring in staphylococcal catheter infection. Irrespective of whether microorganisms ultimately causing clinically apparent catheter infection derive from the patients own microflora or from the hospital environment, and whether the bacteria are of hematogenous origin or from the skin surrounding the catheter wound, the pathogenic events cumulate in a sequence with characteristics common for all kinds of materials, catheter microorganisms and
Institut for Medizinische Mikrobiologie, Westf~lische WilhelmsUniversit&t MOnster, Domagkstr. 10, 48129 M~nster, Germany.
underlying patient conditions (Fig. 1). A clean substratum (i.e., a catheter) is introduced into the vein and subsequently conditioned by host factors adsorbed to the surface. Next, bacterial adhesion occuring leading to colonization of the material. Finally, adhesion is followed by growth of the bacteria on the surface resulting in vertical accumulation of the microorganisms, development of a biofilm, and, clinically, signs and symptoms of catheter infection. In the following, the role of material, host factors, and bacterial properties during these different pathogenic steps will be reviewed.
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•
/ ,,s
,J o
.
•
e
• •
•
4 / /
°/
/
•
o
/
•
• Clean
substratum
Conditioning
film
•
z..'Sm-tO~O 0
Reversible Irreversible Accumulation Adhesion
1. - - Sequence of patogenic events c a u s i n g rent catheter i n f e c t i o n .
Fig.
•
clinically
appa-
Surface Conditioning
After insertion into the bloodstream, the catheter surface is almost immediately covered by plasma proteins, particularly albumin. Albumin is subsequently replaced in a process taking minutes to hours by extracellular matrix proteins and other plasma proROan. Urg., 1994, 3 (3 bis), 355-358
-
3 5 6
-
XII o Conference de Consensus en R~animation et MOdecine d'Urgence
teins, notably flbronectin, fibrinogen and vitronectin, as well as, to various extents, thrombospondin, laminin and collagen. Within 24 to 48 h after insertion, the deposition of flbrinogen/fibrin result in a macroscopically visible fibrin sheath. In addition to proteins, cellular elements, particularly platelets, are deposited on the surface, a phenomenon largely contributing to the blood compatibility. Blood-contacting properties, platelet deposition and activation as well as the adsorption of a variety of adhesive plasma proteins including vitronectin, fibrinogen and fibronectin have been studied using various catheter materials, including polyethylene, silicone rubber, teflon, and polyurethanes. Furthermore, notably polyurethanes have been extensively modified using sulfonate and bioactive peptide grafting, carboxylate and sulphonate ion incorporation, and chemical variation of the hydrophilic soft segment side chains. Finally, physico-chemical properties such as surface hydrophilicity of different materials on blood compatibility has been tested both ex vivo and in vitro. These studies revealed that particular materials, e.g. sulfonated polytetramethyleneoxide polyurethanes, show increased blood compatibility over others, and that vitronectin shows increased adsorption and displacement resistance on the material surface when compared to other plasma proteins [2]. Taken together, in a process of hours to days after insertion of a catheter and as a function of the catheter material used, the surface becomes extensively modified by a complex substratum comprising of cellular and extracellular ligands.
•
Bacterial Adhesion
During the initial adhesion phase, physico-chemical properties of the bacterial cell surface are important and determine whether the cell can attach to the surface. Charged with the same sign, a bacterial cell may approach a surface, attracted by long-range electrostatic and van der Waals forces. This attraction occurs at a distance of 10-20 nm from the surface and results in a reversible adhesion of bacteria which can be readily detached, e.g. by fluid shear forces. Further approach of the microorganisms is inhibited by strong repulsive forces, yet, cells may hover at this point and may adhere more closely to the surface (< i nm) due to a variety of short-range interactions [3]. Staphylococci as well as many catheter materials possess a hydrophobic surface, and, in an aqueous milieu, hydrophobic interactions are among the most important short-range interactions. In a proteinaceous milieu, however, such as plasma, all surfaces are hydrophilic due to albumin adsorption. Therefore, in order to attach irreversibly, bacteria need to possess specific adhesins allowing them to bind to ligands adsorbed on the catheter material. Hence, it is the effect of these short-range forces between ROan. Urg., 1994, 3 (3 bis), 355-358
molecules on the bacterial surface and those on the substratum which is the key to long-term irreversible adhesion. During the last two decades, investigators have characterized the interaction of staphylococcal cells with surface elements on a molecular level. Interestingly, quite different concepts have evolved with respect to S. a u r e u s and CONS. For S. aurens, it is now generally well accepted that the interactions resulting in irreversible adhesion to the conditioned surface are due to specific interactions between S. aureus surface receptors and binding domains in adsorbed proteins. Among these, fibronectin is the protein most extensively studied. It has been shown that fibronectin binds specifically to S. aureus in suspension and that surface-adsorbed fibronectin greatly enhances S. aureus adhesion in a proteinaceous milieu [4, 5]. The binding domain on fibronectin interacting with S. aureus adhesins has been localized on the 27 kD aminoterminal trypsin digest of fibronectin, and two staphylococcal receptors recognizing flbronectin have been cloned and sequenced [6]. Experimental endocarditis models using genetically manipulated, fibronectin-receptor deficient strains as well as animals immunized against the fibronectin receptors have confirmed the importance of the interaction of fibronectin with its receptor on S. aureus in vivo, thus fulfilling the Koch's modified postulates of molecular pathogenesis. The interaction of fibrinogen with a "clumping factor" on S. aureus cells has been used since a long time for microbiological-diagnostic purposes. The role of this receptor in staphylococcal "clumping" using fibrinogen-containing media as well as in the promotion of bacterial adhesion to fibrinogen-coated surfaces has been demonstrated using transposon mutants, and most recently, the staphylococcal fibrinogen receptor has been cloned and sequenced [7]. Interestingly, recent studies using explanted central venous catheters from humans have suggested that fibronectin rather than fibrinogen/fibrin is active in the promotion of S. aureus adhesion to intravascular catheters [8]. The binding of thrombospondin, a major platelet-release protein, to S. aureus has also been demonstrated, and thrombospondin adsorbed to solid surfaces did promote adhesion of all S. aureus and most CoNS strains tested [9]. Interaction with this protein may be of particular relevance at sites of platelet aggregation and thrombus formation. Other plasma and extracellular matrix proteins potentially present on the catheter surface with welldefined binding characteristics to S. aureus include vitronectin and collagen. Contrasting to the large amount of data on blood compatibility of different catheter materials, only a small number of in vitro studies testing staphylococcal adhesion to different materials adsorbed with whole blood or with purified plasma proteins are available. Whole blood-adsorbed polyurethane
X l l ° C o n f 6 r e n c e d e C o n s e n s u s en R ~ a n i m a t i o n et M 6 d e c i n e d ' U r g e n c e - 3 5 7 -
material promoted S. aureus adhesion in an ex vivo shunt model in an order of magnitude less when compared to polyvinylchloride and polyethylene. In an in vitro system determining bacterial attachment and detachment under physiological conditions in realtime, it was shown that S. aureus adhered to uncoated positively charged polyurethanes more readily and less reversible when compared to anionic and nonionic polyurethanes, however, once the different materials were adsorbed with fibrinogen, adherence was virtually identical, i.e. rapid and irreversible. While most researchers have focused on the interaction of S. aureus with soluble host factors, little is known about the interaction of staphylococci with cellular elements. Among blood host ceils, platelets have most often been shown to be deposited on intravenous catheters resulting in formation of microor macrothrombi. However, the role of immobilized platelets in the promotion of bacterial adhesion is currently not well understood. Though it is well accepted that platelets play a pivotal role in infective endocarditis as a nidus for bacterial attachment to the heart valve, little is known about the ligands involved in this interaction, particularly for staphylococci. We recently have demonstrated that in a milieu containing plasma proteins, immobilized platelets greatly promote adhesion of S. aureus and that this interaction is due to a bridging between platelet glycoproteins, fibrinogen/fibrin together with additional unidentified plasma factors, and staphylococcal surface receptors [10]. In another series of experiments, we tested different materials including polymethylmethacrylate, polyethylene, polydimethylsiloxane, sulfonated polyurethanes and phosphated polyurethanes. Interestingly, S. aureus adhesiorr to platelet-adsorbed charged materials (polyurethanes) was significantly higher when compared with other materials, suggesting that on a highly charged surface, platelet damage may result in release of platelet proteins and therefore even greater S. aureus adhesion. In the meantime, other groups have been able to demonstrate a role of immobilized platelets for adhesion of CONS. Whereas the concept of specific interactions of microorganisms with catheter-adsorbed ligands resulting in irreversible adhesion is now well accepted for S. aureus, this is not the case for CONS. Adhesion of clinical CoNS has been shown to be promoted by fibronectin, however, adhesion of CoNS to ex vivo cannula-adsorbed plasma proteins is lower when compared to S. aureus strains. Since in many centers CoNS are contributing more frequently to catheterassociated morbidity, researchers have investigated other mechanisms by which CoNS may specifically interact with catheter surfaces. Two antigens, a "capsular polysaccharide/adhesin" (PS/A) and a "protein adhesin" have been described with adhesion-promoting characterictics, and transposon mutants deficient in PS/A have been constructed
[11]. It should be noted, though, that the adhesion assays the authors used included uncoated catheter material in an albumin- and calcium + + -free milieu and are therefore potentially flawed by the fact that they are measuring modification of hydrophobic interaction rather than interaction of the antigen with surface ligands. On the other hand, evidence from experimental catheter infection models with rabbits passively immunized against PS/A as well as the observation that the majority of clinical CoNS strains was PS/A positive may suggest a role of PS/A in catheter infection. Taking together these findings, it may be concluded that a role particularly of the PS/A antigen under in vivo conditions is possible yet not ultimately proven. Also, in addition to the aforementioned antigens, hemagglutinin(s) have been implicated in adhesion of CoNS to catheter material.
•
Growth and Accumulation
The rapid initial reversible and irreversible adhesion step is followed by a second more prolonged phase of proliferation of the adherent bacteria, accumulation on the polymeric surface, and biofilm formation. This phase is characterized by the ability of staphylococci, particularly of CONS, to grow not only on the polymer surface in the form of large clusters but also three-dimensionally towards the lumen of the catheter [12]. Of importance for this form of growth and accumulation of large amounts of viable bacteria on the surface is the production of an exopolymeric slime substance (ESS). Apparently, ESS is providing the matrix for the inter-cell adhesion necessary for the formation of multiple cell-layers and biofilm formation. In experimental models, ESS formation was Shown to be associated with virulence: Injection of ESS-producing S. epidermidis grown under conditions for accumulative growth into the peritoneal cavity of mice resulted in generalized septicemia and death of the animals in most cases, whereas injection of the same strain, obtained from a liquid culture and washed, did not result in detectable disease [13]. These results are compatible with others obtained with a model of subcutaneous catheter infection. Furthermore, in several clinical studies examining patients with ventriculoperitoneal shunts, chronic ambulatory peritoneal dialysis catheters and nosocomial catheter-related septicemia, pathogenicity of CoNS could be associated with slime production. Thus, several methods have been developed to detect and quantify ESS formation. The composition of ESS produced by CoNS is not clearly defined: Most authors agree that it is a water-soluble, carbohydrate-rich substance consisting of several sugar moieties, teichoic acid, and proteins. However, clear definition of ESS has been hampered in the past due to the dependency of its composition from culturing conditions. R~an. Urg., 1994, 3 (3 bis), 355-358
-
358
-
XII o C o n f e r e n c e de C o n s e n s u s e n R 6 a n i m a t i o n et M 6 d e c i n e d ' U r g e n c e
Two polysaccharid antigens of S. epidermidis have been described, a "slime-associated antigen" with a molecular weight > 50 kD [14], and another polysaccharide antigen apparently strongly associated with accumulative growth of CoNS [15]. Presently it is unclear whether both antigens are of different or of similar composition. Our group has recently generated a mutant CoNS strain retaining its capacity to adhere to a polymer surface but deficient in accumulative growth. Subsequent characterization of the mutant revealed a deficiency in a 115 kD extracellular protein [16]. Currently, the mutant as well as the role of the 115 kD protein in the process of CoNS accumulation and virulence is under investigation.
•
Concluding remarks
It is now clear that the process of catheter colonization and infection is a series of complex events involving material properties, host factors, and bacterial characteristics. Despite considerable progress in our understanding of the pathogenesis of catheter infection, some questions still may appear as intriguingly simple though formidably difficult to resolve: Among the factors adsorbed during conditioning of the surface, which are the ones relevant in vivo? What are the surface characteristics at the site of the cannula wound, i.e., at the interface between unconditioned and conditioned polymer? Do microorganisms sense a nearby surface? What are the differences in the composition of biofilm polysaccharides, characteristics of the bacterial surface and the state of bacterial metabolism between planctonic versus sessile microorganisms? It is to be hoped that answers to some of these and other questions will be available in the near future. Not only will such information be of fundamental interest, but it may also lead to the development of engineered strategies for prevention of catheter-associated infections based on intimate knowledge of the pathogenic factors involved in this process.
References [1] MAKI D.G. - - Infections associated with intravascular lines. In Current clinical topics in infectious diseases. Vol. 3. J.S. Remington and M.N. Swartz, editors. New York, McGrawHill, 1982, 309-363.
mmm
R#an. Urg., 1994, 3 (3 bis), 355-358
[2] PI-FrW.G., WEAVER D.R. and COOPERS.L. - - Fibronectin adsorption kinetics on phase segregated polyurethaneuras. J. Biomater. Sci. Polym. Ed., 1993, 4, 337-346. [3] ALLISON D.G. - - Biofilm-associated exopolysaccharides. Microbiology Europe, 1993, 16-19. [4] PROCTORR.A., MOSHER D.F. and OLBRANTZP.J. - - Fibronectin binding to Staphylococcus aureus. J. BioL Chem., 1982, 257, 14788-14794. [5] HERRMANNM., VAUDAUX P., PITrET D., AUCKENTHALERR., LEW P.D., SCHUMACRER-PERDREAUF., PETERS G. and WALDVOGEL F.A. - - Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material. J. Infect. Dis., 1988, 158, 693-701. [6] JONSSON K., SIGNAS C., MOLLER H.P. and LINDBERG M. Two different genes encode fibronectin-binding proteins in Staphylococcus aureus The complet nucleotide sequence and characterization of the second gene. Eur. J. Biochem, 1991, 202, 1041-1048. [7] MCDEVWF D., FRAN~;OISP., VAUDAUX P. and FOSTERT.J. - Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. MoL Microbiol., 1994, (In Press). [8] VAUDAUXP., PI'FrET D., HAEBERLIA., LERCH P., MORGENTRALER J.-J., PROCTOR R.A., WALDVOGEL F.A. and LEW P.D. - Fibrinonectin is more active than fibrin or fibrinogen in promoting Staphylococcus aureus adherence to inserted intravascular catheters. J. Infect. Dis., 1993, 167, 633-641. [9] HERRMANN M., SUCHARD S.J., BOXER L.A., WALDVOGEL F.A. and LEW P.D. - - Thrombospondin binds to Staphylococcus aureusand promotes staphylocollal adherence to surfaces. Infect. Immun., 1991, 59, 279-288. [10] HERRMANN M., LAI Q.J., ALBRECHT R.M., MOSHER D.F. and PROCTOR A. - - Adhesion of Staphylococcus aureus to surface-bound platelets: Role of fibrinogen/fibrin and platelet integrins. J. Infect. Dis., 1993, 167, 312-322. [11] MULLER E., HUBNER J., GUTTIEREZ N., TAKEDA S., GOLDMAN D.A. and PIER G.D. - - Isolation and characterization of transposon mutants of Staphylococcus epidermidis deficient in capsular polysaccharid/adhesion and slime. Infect. Immun., 1993, 61, 551-558. [12] PETERSG., LOCCl R. and PULVERERG. - - Microbial adhesion of prosthetic devices. I1. Scanning electron microscopy of naturally infected intravenous catheters. ZbL Bakt. Hyg. I. Abt. Orig. B., 1981, 173, 293-299. [13] PETER8 G., SCHUMACHER-PERDREAU F., JANSEN B., BEY M. and PULVERER G. - - Biology of Staphylococcus epidermidis extracellular slime, in: Pathogenicity and clinical significance of coagulase-negative staphylococci. G. Puiverer, P.G. QuiP and G. Peters, editors. Gustav Fischer Verlag, Stuttgart, New York, 1987, 15-32. [14] CHRISTENSENG.D., BARKER L.P., MAWHINNEYT.P., BADDOUR L.M. and SIMPSONW. - - Identification of an antigenic marker of slime production for Staphylococcus epidermidis. Infect. Immun., 1990, 58, 2906-2911. [15] MACK D., SIEMSSENN., LAUFS R. - - Parallel induction by glucose of ahderence and a polysaccharide antigen specific for plastic-adherent Staphylococcus epidermidis. Infect. Immun. 1992, 60 : 2048-2057. [16] SCHUMACHER-PERDREAUF., HEILMANNC., PETERS G., GOTZ F. and PULVERERG. - - Comparative analysis of a biofilm forming Staphylococcus epidermidisstrain and its accumulationnegative isogenic mutant. FEMS MicrobioL Lett. (In Press), 1994. -
-
•
Introduction
Central venous catheters are indispensable in modern intensive care, since they are used for a large variety of purposes, including administration of fluids and electrolytes, blood products, drugs, for total parenteral nutrition, and for hemodynamic monitoring. Polyurethanes and polyvinylchloride are among the most frequently used materials for these devices. However, microbial colonization of intravenous lines is the single most important source for nosocomial bacteremia with its potential consequences of sepsis and metastic infection. It contributes significantly to hospital-associated morbidity and mortality. Although bacteria such as Pseudomonas, other nonfermenting gram-negative rods, enterobacteriaceae, diphtheroids and enterococci as well as fungi are causing intravenous catheter infection, staphylococci, particularly coagulase-negative staphylococci (CONS) but also S. aureus, are by far the most important since most frequently isolated strains [1]. In the past, interactions between material and bacteria have been most extensively studied for staphylococci, and although many of the pathogenic concepts in catheter infection may also be valid for other bacterial species, this contribution will focus on the pathogenic events occuring in staphylococcal catheter infection. Irrespective of whether microorganisms ultimately causing clinically apparent catheter infection derive from the patients own microflora or from the hospital environment, and whether the bacteria are of hematogenous origin or from the skin surrounding the catheter wound, the pathogenic events cumulate in a sequence with characteristics common for all kinds of materials, catheter microorganisms and
Institut for Medizinische Mikrobiologie, Westf~lische WilhelmsUniversit&t MOnster, Domagkstr. 10, 48129 M~nster, Germany.
underlying patient conditions (Fig. 1). A clean substratum (i.e., a catheter) is introduced into the vein and subsequently conditioned by host factors adsorbed to the surface. Next, bacterial adhesion occuring leading to colonization of the material. Finally, adhesion is followed by growth of the bacteria on the surface resulting in vertical accumulation of the microorganisms, development of a biofilm, and, clinically, signs and symptoms of catheter infection. In the following, the role of material, host factors, and bacterial properties during these different pathogenic steps will be reviewed.
/ •
d
,-'
•
/ ,,s
,J o
.
•
e
• •
•
4 / /
°/
/
•
o
/
•
• Clean
substratum
Conditioning
film
•
z..'Sm-tO~O 0
Reversible Irreversible Accumulation Adhesion
1. - - Sequence of patogenic events c a u s i n g rent catheter i n f e c t i o n .
Fig.
•
clinically
appa-
Surface Conditioning
After insertion into the bloodstream, the catheter surface is almost immediately covered by plasma proteins, particularly albumin. Albumin is subsequently replaced in a process taking minutes to hours by extracellular matrix proteins and other plasma proROan. Urg., 1994, 3 (3 bis), 355-358
-
3 5 6
-
XII o Conference de Consensus en R~animation et MOdecine d'Urgence
teins, notably flbronectin, fibrinogen and vitronectin, as well as, to various extents, thrombospondin, laminin and collagen. Within 24 to 48 h after insertion, the deposition of flbrinogen/fibrin result in a macroscopically visible fibrin sheath. In addition to proteins, cellular elements, particularly platelets, are deposited on the surface, a phenomenon largely contributing to the blood compatibility. Blood-contacting properties, platelet deposition and activation as well as the adsorption of a variety of adhesive plasma proteins including vitronectin, fibrinogen and fibronectin have been studied using various catheter materials, including polyethylene, silicone rubber, teflon, and polyurethanes. Furthermore, notably polyurethanes have been extensively modified using sulfonate and bioactive peptide grafting, carboxylate and sulphonate ion incorporation, and chemical variation of the hydrophilic soft segment side chains. Finally, physico-chemical properties such as surface hydrophilicity of different materials on blood compatibility has been tested both ex vivo and in vitro. These studies revealed that particular materials, e.g. sulfonated polytetramethyleneoxide polyurethanes, show increased blood compatibility over others, and that vitronectin shows increased adsorption and displacement resistance on the material surface when compared to other plasma proteins [2]. Taken together, in a process of hours to days after insertion of a catheter and as a function of the catheter material used, the surface becomes extensively modified by a complex substratum comprising of cellular and extracellular ligands.
•
Bacterial Adhesion
During the initial adhesion phase, physico-chemical properties of the bacterial cell surface are important and determine whether the cell can attach to the surface. Charged with the same sign, a bacterial cell may approach a surface, attracted by long-range electrostatic and van der Waals forces. This attraction occurs at a distance of 10-20 nm from the surface and results in a reversible adhesion of bacteria which can be readily detached, e.g. by fluid shear forces. Further approach of the microorganisms is inhibited by strong repulsive forces, yet, cells may hover at this point and may adhere more closely to the surface (< i nm) due to a variety of short-range interactions [3]. Staphylococci as well as many catheter materials possess a hydrophobic surface, and, in an aqueous milieu, hydrophobic interactions are among the most important short-range interactions. In a proteinaceous milieu, however, such as plasma, all surfaces are hydrophilic due to albumin adsorption. Therefore, in order to attach irreversibly, bacteria need to possess specific adhesins allowing them to bind to ligands adsorbed on the catheter material. Hence, it is the effect of these short-range forces between ROan. Urg., 1994, 3 (3 bis), 355-358
molecules on the bacterial surface and those on the substratum which is the key to long-term irreversible adhesion. During the last two decades, investigators have characterized the interaction of staphylococcal cells with surface elements on a molecular level. Interestingly, quite different concepts have evolved with respect to S. a u r e u s and CONS. For S. aurens, it is now generally well accepted that the interactions resulting in irreversible adhesion to the conditioned surface are due to specific interactions between S. aureus surface receptors and binding domains in adsorbed proteins. Among these, fibronectin is the protein most extensively studied. It has been shown that fibronectin binds specifically to S. aureus in suspension and that surface-adsorbed fibronectin greatly enhances S. aureus adhesion in a proteinaceous milieu [4, 5]. The binding domain on fibronectin interacting with S. aureus adhesins has been localized on the 27 kD aminoterminal trypsin digest of fibronectin, and two staphylococcal receptors recognizing flbronectin have been cloned and sequenced [6]. Experimental endocarditis models using genetically manipulated, fibronectin-receptor deficient strains as well as animals immunized against the fibronectin receptors have confirmed the importance of the interaction of fibronectin with its receptor on S. aureus in vivo, thus fulfilling the Koch's modified postulates of molecular pathogenesis. The interaction of fibrinogen with a "clumping factor" on S. aureus cells has been used since a long time for microbiological-diagnostic purposes. The role of this receptor in staphylococcal "clumping" using fibrinogen-containing media as well as in the promotion of bacterial adhesion to fibrinogen-coated surfaces has been demonstrated using transposon mutants, and most recently, the staphylococcal fibrinogen receptor has been cloned and sequenced [7]. Interestingly, recent studies using explanted central venous catheters from humans have suggested that fibronectin rather than fibrinogen/fibrin is active in the promotion of S. aureus adhesion to intravascular catheters [8]. The binding of thrombospondin, a major platelet-release protein, to S. aureus has also been demonstrated, and thrombospondin adsorbed to solid surfaces did promote adhesion of all S. aureus and most CoNS strains tested [9]. Interaction with this protein may be of particular relevance at sites of platelet aggregation and thrombus formation. Other plasma and extracellular matrix proteins potentially present on the catheter surface with welldefined binding characteristics to S. aureus include vitronectin and collagen. Contrasting to the large amount of data on blood compatibility of different catheter materials, only a small number of in vitro studies testing staphylococcal adhesion to different materials adsorbed with whole blood or with purified plasma proteins are available. Whole blood-adsorbed polyurethane
X l l ° C o n f 6 r e n c e d e C o n s e n s u s en R ~ a n i m a t i o n et M 6 d e c i n e d ' U r g e n c e - 3 5 7 -
material promoted S. aureus adhesion in an ex vivo shunt model in an order of magnitude less when compared to polyvinylchloride and polyethylene. In an in vitro system determining bacterial attachment and detachment under physiological conditions in realtime, it was shown that S. aureus adhered to uncoated positively charged polyurethanes more readily and less reversible when compared to anionic and nonionic polyurethanes, however, once the different materials were adsorbed with fibrinogen, adherence was virtually identical, i.e. rapid and irreversible. While most researchers have focused on the interaction of S. aureus with soluble host factors, little is known about the interaction of staphylococci with cellular elements. Among blood host ceils, platelets have most often been shown to be deposited on intravenous catheters resulting in formation of microor macrothrombi. However, the role of immobilized platelets in the promotion of bacterial adhesion is currently not well understood. Though it is well accepted that platelets play a pivotal role in infective endocarditis as a nidus for bacterial attachment to the heart valve, little is known about the ligands involved in this interaction, particularly for staphylococci. We recently have demonstrated that in a milieu containing plasma proteins, immobilized platelets greatly promote adhesion of S. aureus and that this interaction is due to a bridging between platelet glycoproteins, fibrinogen/fibrin together with additional unidentified plasma factors, and staphylococcal surface receptors [10]. In another series of experiments, we tested different materials including polymethylmethacrylate, polyethylene, polydimethylsiloxane, sulfonated polyurethanes and phosphated polyurethanes. Interestingly, S. aureus adhesiorr to platelet-adsorbed charged materials (polyurethanes) was significantly higher when compared with other materials, suggesting that on a highly charged surface, platelet damage may result in release of platelet proteins and therefore even greater S. aureus adhesion. In the meantime, other groups have been able to demonstrate a role of immobilized platelets for adhesion of CONS. Whereas the concept of specific interactions of microorganisms with catheter-adsorbed ligands resulting in irreversible adhesion is now well accepted for S. aureus, this is not the case for CONS. Adhesion of clinical CoNS has been shown to be promoted by fibronectin, however, adhesion of CoNS to ex vivo cannula-adsorbed plasma proteins is lower when compared to S. aureus strains. Since in many centers CoNS are contributing more frequently to catheterassociated morbidity, researchers have investigated other mechanisms by which CoNS may specifically interact with catheter surfaces. Two antigens, a "capsular polysaccharide/adhesin" (PS/A) and a "protein adhesin" have been described with adhesion-promoting characterictics, and transposon mutants deficient in PS/A have been constructed
[11]. It should be noted, though, that the adhesion assays the authors used included uncoated catheter material in an albumin- and calcium + + -free milieu and are therefore potentially flawed by the fact that they are measuring modification of hydrophobic interaction rather than interaction of the antigen with surface ligands. On the other hand, evidence from experimental catheter infection models with rabbits passively immunized against PS/A as well as the observation that the majority of clinical CoNS strains was PS/A positive may suggest a role of PS/A in catheter infection. Taking together these findings, it may be concluded that a role particularly of the PS/A antigen under in vivo conditions is possible yet not ultimately proven. Also, in addition to the aforementioned antigens, hemagglutinin(s) have been implicated in adhesion of CoNS to catheter material.
•
Growth and Accumulation
The rapid initial reversible and irreversible adhesion step is followed by a second more prolonged phase of proliferation of the adherent bacteria, accumulation on the polymeric surface, and biofilm formation. This phase is characterized by the ability of staphylococci, particularly of CONS, to grow not only on the polymer surface in the form of large clusters but also three-dimensionally towards the lumen of the catheter [12]. Of importance for this form of growth and accumulation of large amounts of viable bacteria on the surface is the production of an exopolymeric slime substance (ESS). Apparently, ESS is providing the matrix for the inter-cell adhesion necessary for the formation of multiple cell-layers and biofilm formation. In experimental models, ESS formation was Shown to be associated with virulence: Injection of ESS-producing S. epidermidis grown under conditions for accumulative growth into the peritoneal cavity of mice resulted in generalized septicemia and death of the animals in most cases, whereas injection of the same strain, obtained from a liquid culture and washed, did not result in detectable disease [13]. These results are compatible with others obtained with a model of subcutaneous catheter infection. Furthermore, in several clinical studies examining patients with ventriculoperitoneal shunts, chronic ambulatory peritoneal dialysis catheters and nosocomial catheter-related septicemia, pathogenicity of CoNS could be associated with slime production. Thus, several methods have been developed to detect and quantify ESS formation. The composition of ESS produced by CoNS is not clearly defined: Most authors agree that it is a water-soluble, carbohydrate-rich substance consisting of several sugar moieties, teichoic acid, and proteins. However, clear definition of ESS has been hampered in the past due to the dependency of its composition from culturing conditions. R~an. Urg., 1994, 3 (3 bis), 355-358
-
358
-
XII o C o n f e r e n c e de C o n s e n s u s e n R 6 a n i m a t i o n et M 6 d e c i n e d ' U r g e n c e
Two polysaccharid antigens of S. epidermidis have been described, a "slime-associated antigen" with a molecular weight > 50 kD [14], and another polysaccharide antigen apparently strongly associated with accumulative growth of CoNS [15]. Presently it is unclear whether both antigens are of different or of similar composition. Our group has recently generated a mutant CoNS strain retaining its capacity to adhere to a polymer surface but deficient in accumulative growth. Subsequent characterization of the mutant revealed a deficiency in a 115 kD extracellular protein [16]. Currently, the mutant as well as the role of the 115 kD protein in the process of CoNS accumulation and virulence is under investigation.
•
Concluding remarks
It is now clear that the process of catheter colonization and infection is a series of complex events involving material properties, host factors, and bacterial characteristics. Despite considerable progress in our understanding of the pathogenesis of catheter infection, some questions still may appear as intriguingly simple though formidably difficult to resolve: Among the factors adsorbed during conditioning of the surface, which are the ones relevant in vivo? What are the surface characteristics at the site of the cannula wound, i.e., at the interface between unconditioned and conditioned polymer? Do microorganisms sense a nearby surface? What are the differences in the composition of biofilm polysaccharides, characteristics of the bacterial surface and the state of bacterial metabolism between planctonic versus sessile microorganisms? It is to be hoped that answers to some of these and other questions will be available in the near future. Not only will such information be of fundamental interest, but it may also lead to the development of engineered strategies for prevention of catheter-associated infections based on intimate knowledge of the pathogenic factors involved in this process.
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