Propionibacterium acnes in the etiology of endocarditis]

Propionibacterium acnes is the gram positive anaerobic bacteria belongs to the normal skin and oral microbial flora. The participation of this microorganism in the infective endocarditis is still controversial. The aim of the study was to perform the diagnostic and therapeutic difficulties in 5 patients with infective endocarditis caused by Propionibacterium acnes. In 3 out of 5 patients the infective endocarditis developed after prosthesis valve replacement, in 2 others on the native valves. The inserted prostheses were mechanical ones, propionibacterium acnes was identified as causative organisms in all of the causes (two positive blood and/or valve culture). The bacterial strains were sensitive to the antibiotics as: penicillins, cephalosporins, clindamycin, and vancomycin, however cephalosporins used at the beginning of the treatment in 3 patients and clindamycin in 1 patient had limited clinical efficacy. Later treatment with timentin, augmentin and tienamycin was successful in 3 patients; one patient was cured with vancomycin. One patient died because of septic, embolic complication in early stage of illness. We conclude the effectiveness of penicillins in combination with clavulanic acid and tienamycin in therapy of infective endocarditis due to Propionibacterium acnes. The treatment should be lasted during 4-6 weeks.     

Platelet receptor polymorphisms do not influence Staphylococcus aureus-platelet interactions or infective endocarditis

Cardiac vegetations result from bacterium-platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen Pl(A1/A2) and FcγRIIa H131R genotype of healthy volunteers (n?=?160) and patients with infective endocarditis (n?=?40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus-platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P?>?0.05 for all), even though patients with the GPIIIa Pl(A1/A1) genotype had increased in vivo platelet activation (P?=?0.001). Furthermore, neither GPIIIa Pl(A1/A2) nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P?>?0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus-platelet interactions in vitro or the clinical course of infective endocarditis.     

Prosthetic valve endocarditis.

During 1965 to 1982, 32 episodes of infective endocarditis on prosthetic valves in 30 patients were treated at this hospital. In early endocarditis (presenting within four months of operation) staphylococci were the organisms most commonly responsible. Early endocarditis appears to be declining in incidence and is largely preventable; sternal sepsis was the main predisposing factor, requiring urgent and effective treatment. Streptococci were the most common organisms in late onset disease, but as with natural valve endocarditis a wide range or organisms was responsible. All but one of the patients with early onset disease were treated conservatively, but mortality was high; prompt surgical replacement of infected prostheses is probably indicated in such patients. Medical management was effective in most patients with late onset disease, and for them early surgical intervention may not be justified.     

Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix

A range of Streptococcus bacteria are able to interact with blood platelets to form a thrombus (clot). Streptococcus gordonii is ubiquitous within the human oral cavity and amongst the common pathogens isolated from subjects with infective endocarditis. Two cell surface proteins, Hsa and Platelet adherence protein A (PadA), in S. gordonii mediate adherence and activation of platelets. In this study, we demonstrate that PadA binds activated platelets and that an NGR (Asparagine‐Glycine‐Arginine) motif within a 657 amino acid residue N‐terminal fragment of PadA is responsible for this, together with two other integrin‐like recognition motifs RGT and AGD. PadA also acts in concert with Hsa to mediate binding of S. gordonii to cellular fibronectin and vitronectin, and to promote formation of biofilms. Evidence is presented that PadA and Hsa are each reliant on the other's active presentation on the bacterial cell surface, suggesting cooperativity in functions impacting both colonization and pathogenesis.     

Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin-binding proteins FnBPA and FnBPB are major platelet-activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen-binding A domain or the fibronectin-binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA-promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV-3) specific for the FcgammaRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcgammaRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV-3 suggesting that the IgG-FcgammaRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.     

Binding of laminin to oral and endocarditis strains of viridans streptococci.

Attachment of bacteria to the host tissue is regarded as a crucial step in the development of many types of infections. Recent studies by us and others have shown that matrix proteins which serve as adhesion proteins for eucaryotic cells may also be recognized by some bacteria. In the present communication, we report that several strains of viridans streptococci are able to bind to laminin. Most strains isolated from blood and heart valves of patients with endocarditis expressed laminin receptors, whereas only a few of the strains isolated from the oral cavity recognized this protein. This observation indicates that laminin binding might be an important factor in the pathogenesis of viridans endocarditis. Laminin binding to two strains (Streptococcus mitis UAB594 and UAB597) isolated from patients with endocarditis was characterized further. The bacterial cells expressed a limited number of laminin receptors (4 X 10(2) to 1 X 10(3) per cell) which bound the protein in a high-affinity interaction (Kd, 40 to 80 nM). This receptor of S. mitis UAB594 was heat labile and could be solubilized from bacteria by brief digestion with trypsin. Solubilized receptors which competed with cell-bound receptors for 125I-laminin could be adsorbed on laminin-Sepharose but not on Sepharose substituted with fibrinogen or fibronectin. Comparison of laminin receptors from S. mitis with those previously described for Streptococcus pyogenes suggest that different sites in the laminin molecule are recognized by the two bacteria and hence that the corresponding receptor molecules are not identical.     

Screening of Peptides that Inhibit Bacterial Binding to Fibronectin using Combinatorial Peptide Libraries

Infective endocarditis is often caused by passage of oral endogenous bacteria into the blood stream. Such bacteremia occurs after tooth extraction, and occasionally even after brushing of the teeth. Abnormal or damaged heart valves, including artificial valves, show high risk of infection. Antibiotics are widely used to prevent this infection, however, frequent use of these have resulted in the generation of resistant mutants, which generate serious social problems. Thus, the development of more effective and safer drugs for the prevention of such infections is very desirable. The adhesion of bacteria to fibronectin, one of the major extracellular matrix (ECM) protein exposed on the wound endocardia, is considered critical for the infection. We have previously found a novel mode of interaction between endocarditis-causing bacteria and human fibronectin. The present study focuses on the discovery of candidate compounds that inhibit the association between microorganisms and fibronectin. Positional scanning libraries (PSL) with N-terminal biotinylated 6-mer peptides have been constructed and screened for binding to a monoclonal antibody for fibronectin that inhibits the bacterial fibronectin-binding. The consensus sequences derived from these experiments are expected to be structural mimetics of the local structure of fibronectin involved in the bacterial adhesion. Since individual synthetic 6-mer peptides did not show the desired action, discontinuous epitopes can be envisaged and therefore a 9-mer-PSL was constructed to reveal conformational epitopes. In the second library, several 9-mer peptides based on the screening were synthesized and gave improved results.Australian Peptide Conference Issue     

Inhibition of fibronectin binding of some bacterial cells by subtle pH increase within the physiological range

The fibronectin (Fn)-binding ability of microorganisms is considered to be involved in their pathogenicities. Granulicatella adiacens, a member of the oral flora and a causative agent of culture-negative infective endocarditis, showed nearly maximum binding to immobilized Fn at pH 7.2 but greatly reduced binding at a slightly higher pH 7.4 and almost no binding at pH 7.6 in the presence of physiological concentration of NaCl (0.15 M). A similar pH-sensitive Fn-binding property was noted with Escherichia coli and Abiotrophia defectiva, but not with Streptococcus pyogenes nor Staphylococcus aureus. In contrast, bindings to laminin and fibrinogen observed for some of these strains were unaffected by the same pH changes. This fastidious pH-dependency of Fn-binding abilities of some bacteria warns that the pH condition must be seriously considered in the in vitro assay of bacterial adherence to fibronectin.     

血链球菌与疾病研究进展

血链球菌是早期定植在口腔内的细菌之一,也是口腔内的常驻菌.血链球菌作为牙周有益菌对大多数牙周可疑致病菌具有拮抗作用.其主要机制为产生过氧化氢和血链素.由于各种原因进入血液循环后可引起感染性心内膜炎,主要机制为血链球菌的表面抗原使血小板产生黏附、聚集,其在血小板表面的结合位点在血小板膜糖蛋白Ib附近.单核细胞在血链球菌的刺激下表达大量的组织因子,激活外源性凝血途径形成血栓.     

The role of endothelial cell biology in endocarditis

The treatment of endocarditis remains a challenge for physicians, even in times of modern antibiotic treatment. Depending on its cause, endocarditis can either be of infectious or non-infectious origin. Infective endocarditis is caused by bacterial (or fungal) pathogens, and the clinical course is critically dependent on the virulence factors of the specific microorganisms involved. Therefore, the clinical type of endocarditis can be divided into an acute and more aggressive form and a subacute form (endocarditis lenta). Much of our knowledge regarding the pathogenesis of infective endocarditis is based on studies of the virulence of Staphylococcus aureus, which has become the most frequent cause of infective endocarditis nowadays. However, independently of the underlying cause of endocarditis (infectious or noninfectious), the pathogenesis involves the damage and disturbance of endothelial function and the formation of associated “vegetation”. Surprisingly little is known about the specific role of the endothelium in the pathogenesis of endocarditis. This review will thus give insights into current knowledge of the pathogenesis of endocarditis with a focus on the role of the endothelium.     

The structure of fibronectin and its role in cellular adhesion

Fibronectin is a large, adhesive glycoprotein which is found in a number of locations, most notably on cell surfaces, in extracellular matrixes, and in blood. Fibronectin has been detected in all vertebrates tested and in many invertebrates. Its presence in sponges is significant because this suggests that fibronectin may have appeared very early in evolution, possibly with the most primitive multicellular organisms. Cellular and plasma fibronectins have many striking similarities. However, the locations of the polypcptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronectin by means of simple post-translational proteolysis. Instead, these different types of fibronectin may be products of different genes or of differentially spliced messenger RNA molecules. Amniotic fluid fibronectin is possibly a third form of the protein. Cellular and plasma fibronectins are composed of at least six protcaseresistant domains which contain specific binding sites for actin, gelatin, heparin, Staphylococcus aureus, transglutarninase, fibrin, DNA, and a cell surface receptor. The relative locations of these domains have been mapped in the primary structure of fibronectin. The cell surface receptor for fibronectin has not been positively identified, but may be a glycoprotein, a glycolipid, or a complex of the two. Although cell-substratum adhesion is mediated by fibronectin, the locations of the areas of closest approach of the cell to the substratum (the adhesion plaques) and fibronectin are not coincident under conditions of active cell growth. Under conditions of cell growth arrest in low scrum concentrations, some fibronectin may become localized at the adhesion plaques. Models describing the domain structure of fibronectin and the molecular organization of the adhesion plaque area are presented.     

Production of cytokines by monocytes, epithelial and endothelial cells activated by Streptococcus bovis

There are numerous reports documenting the correlation between Streptococcus bovis bacteraemia and endocarditis in conjunction with colonic diseases. The adherence of S. bovis to either buccal or intestinal epithelial cells seems to be the initial process in colonization and subsequent infection of the host, allowing further adhesion of S. bovis to either endothelial cells or extracellular matrix components which leads to infective endocarditis. Bacterial entry at tumour sites is further assisted by the local action of cytokines that promotes vasodilatation and increased capillary permeability. Thus the ability of S. bovis to adhere to and to stimulate human cells may contribute to the pathogenicity of this bacteria. In the present study, we have shown the ability of S. bovis and wall-extracted antigens (WEA) to adhere to human buccal (KB) or intestinal (Caco-2) epithelial cell lines, to human saphenous vein endothelial cells, to human monocytic cell line (THP-1) and to extracellular matrix components (ECM) (fibronectin, collagen and laminin). The fixation of S. bovis on cells was followed by the synthesis of IL-8 from all the cells except Caco-2, whereas S. bovis WEA was able to induce cytokine synthesis from all of them, showing the immunomodulatory effect of S. bovis and S. bovis WEA on different cells.     

Adhesion of Enterotoxigenic (ETEC) and Bovine Mastitis Escherichia coli Strains to Rat Embryonic Fibroblasts: Role of Amino-Terminal Domain of Fibronectin in Bacterial Adhesion

Adherence of human enterotoxigenic and bovine mastitis Escherichia coli to rat embryonic fibroblasts was studied. Adhesion of E. coli strains B34289c (human) and 1407 (bovine) was rapid and reached maximum after 30–40 min. Strain 1410 (bovine), which binds fibronectin but not its 29K amino-terminal fragment, did not adhere to the fibroblasts. Strain B34289c grown at 25 C or below and at 40 C or above lost its binding and adhesive properties simultaneously. Maximum binding and adhesion for this strain was achieved when it was grown at 33 C. Strains grown at this temperature adsorbed to fibronectin-, 29K fragment-, and Octyl Sepharose, with the exception of bovine strain 1410, which did not adsorb to 29K-Sepharose as expected. None of the strains adsorbed to cross-linked Sepharose 4B. 29K-IgG and Fab fragments thereof specifically blocked both binding (max 55%) and adhesion (>95%). Sonicated and trypsin-treated bacteria were no longer able to bind or adhere. The supernatant of sonicated bacteria inhibited both binding and adhesion. Penicillin G at 0.5 μg/ml (1/5 minimal inhibitory concentration: MIC) and tetracycline at 0.2 μg/ml (1/5 MIC), when included in the growth medium, suppressed the cell surface components responsible for fibronectin binding and fibroblast adhesion. The presence of fibronectin was demonstrated in the fibroblast extracellular matrix by immunofluorescens with 29K-IgG antibodies.     

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.     

LOX-1 supports adhesion of Gram-positive and Gram-negative bacteria

Adhesion of bacteria to vascular endothelial cells as well as mucosal cells and epithelial cells appears to be one of the initial steps in the process of bacterial infection, including infective endocarditis. We examined whether lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), a member of scavenger receptor family molecules with C-type lectin-like structure, can support adhesion of Gram-positive and Gram-negative bacteria. Chinese hamster ovary-K1 (CHO-K1) cells stably expressing LOX-1 can support binding of FITC-labeled Staphylococcus aureus and Escherichia coli, which was suppressed by poly(I) and an anti-LOX-1 mAb. Adhesion of these bacteria to LOX-1 does not require divalent cations or serum factors and can be supported under both static and nonstatic conditions. Cultured bovine aortic endothelial cells (BAEC) can also support adhesion of FITC-labeled S. aureus, which was similarly suppressed by poly(I) and an anti-LOX-1 mAb. In contrast, binding of FITC-labeled E. coli to BAEC was partially inhibited by the anti-LOX-1 mAb, and poly(I) did not block FITC-labeled E. coli adhesion to BAEC, but, rather, enhanced it under a static condition. TNF-alpha increased LOX-1-dependent adhesion of E. coli, but not that of S. aureus, suggesting that S. aureus adhesion to BAEC may require additional molecules, which cooperate with LOX-1 and suppressed by TNF-alpha. Taken together, LOX-1 can work as a cell surface receptor for Gram-positive and Gram-negative bacteria, such as S. aureus and E. coli, in a mechanism similar to that of class A scavenger receptors; however, other unknown molecules may also be involved in the adhesion of E. coli to BAEC, which is enhanced by poly(I).     

Fluorescence-based bacterial overlay method for simultaneous in situ quantification of surface-attached bacteria

For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.     

Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis Is Facilitated by the Multiple Repeats within FnBPA

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α₅β₁ integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.     

The role of fibronectin in the pathogenesis of meningococcal infection

We have characterized an interaction of 20 strains of Neisseria meningitidis serogroups A, B, C, 29E, W-135 and Z with immobilized fibronectin of human plasma. The adhesion of meningococci to fibronectin was determined by the extent of piliated cells and did not depend on the meningococcal serogroup. Binding of non-piliated or weakly piliated strains (2-5% of piliated cells in the stock) was sufficiently greater than those piliated (8-10%), where the adhesion to fibronectin was not at all observed. The examination of two well-piliated strains showed that the loss of pili resulted in the increase of bacterial adhesion to fibronectin. Constants of association and dissociation of piliated and non-piliated strains to fibronectin were calculated. The role of meningococci-fibronectin interaction in the pathogenesis of meningococcal infection is discussed.     

Platelet aggregation by oral streptococci

One proposed mechanism in the pathogenesis of infective endocarditis is the direct aggregation of platelets by the bacteria causing the disease. Some, but not all, strains of Streptococcus sanguis have been reported to aggregate platelets but the taxonomy of this and related taxa has changed recently. The ability to aggregate platelets by 24 genetically grouped laboratory stock strains was studied along with 8 recent isolates from cases of endocarditis. Strains belonging to S. sanguis could aggregate platelets, but not S. gordonii, "S. parasanguis", S. mitis, S. oralis or related taxa. Also, preliminary data indicate that certain biotypes of S. sanguis lack the ability to aggregate platelets. Of the recent clinical isolates, only 4 aggregated platelets and each of these showed phenotypes typical of S. sanguis. These data suggest that the ability to aggregate platelets is not essential for an organism to be able to cause endocarditis, although it may be a significant virulence factor.