Tiago Rafael Veloso

Induction of Experimental Endocarditis by Continuous Low-Grade Bacteremia Mimicking Spontaneous Bacteremia in Humans

ABSTRACT Transient high-grade bacteremia following invasive procedures carries a risk of infective endocarditis (IE). This is supported by experimental endocarditis. On the other hand, case-control studies showed that IE could be caused by cumulative exposure to low-grade bacteremia occurring during daily activities. However, no experimental demonstration of this latter possibility exists. This study investigated the infectivity in animals of continuous low-grade bacteremia compared to that of brief high-grade bacteremia. Rats with aortic vegetations were inoculated with Streptococcus intermedius, Streptococcus gordonii or Staphylococcus aureus (strains Newman and P8). Animals were challenged with 103 to 106 CFU. Identical bacterial numbers were given by bolus (1 ml in 1 min) or continuous infusion (0.0017 ml/min over 10 h). Bacteremia was 50 to 1,000 times greater after bolus than during continuous inoculation. Streptococcal bolus inoculation of 105 CFU infected 63 to 100% vegetations compared to 30 to 71% infection after continuous infusion (P > 0.05). When increasing the inoculum to 106 CFU, bolus inoculation infected 100% vegetations and continuous infusion 70 to 100% (P > 0.05). S. aureus bolus injection of 103 CFU infected 46 to 57% valves. This was similar to the 53 to 57% infection rates produced by continuous infusion (P > 0.05). Inoculation of 104 CFU of S. aureus infected 80 to 100% vegetations after bolus and 60 to 75% after continuous infusion (P > 0.05). These results show that high-level bacteremia is not required to induce experimental endocarditis and support the hypothesis that cumulative exposure to low-grade bacteremia represents a genuine risk of IE in humans.

Use of a Human-Like Low-Grade Bacteremia Model of Experimental Endocarditis To Study the Role of Staphylococcus aureus Adhesins and Platelet Aggregation in Early Endocarditis

ABSTRACT Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 106 CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

Prophylaxis of Experimental Endocarditis with Antiplatelet and Antithrombin Agents: a Role for Long-term Prevention of Infective Endocarditis in Humans?

BACKGROUND Infective endocarditis (IE) mostly occurs after spontaneous low-grade bacteremia. Thus, IE cannot be prevented by circumstantial antibiotic prophylaxis. Platelet activation following bacterial-fibrinogen interaction or thrombin-mediated fibrinogen-fibrin polymerization is a critical step in vegetation formation. We tested the efficacy of antiplatelet and antithrombin to prevent experimental IE. METHODS A rat model of experimental IE following prolonged low-grade bacteremia mimicking smoldering bacteremia in humans was used. Prophylaxis with antiplatelets (aspirin, ticlopidine [alone or in combination], eptifibatide, or abciximab) or anticoagulants (antithrombin dabigatran etexilate or anti-vitamin K acenocoumarol) was started 2 days before inoculation with Streptococcus gordonii or Staphylococcus aureus. Valve infection was assessed 24 hours later. RESULTS Aspirin plus ticlopidine, as well as abciximab, protected 45%-88% of animals against S. gordonii and S. aureus IE (P < .05). Dabigatran etexilate protected 75% of rats against IE due to S. aureus (P < .005) but failed to protect against S. gordonii (<30% protection). Acenocoumarol was ineffective. CONCLUSIONS Antiplatelet and direct antithrombin agents may be useful in the prophylaxis of IE in humans. In particular, the potential dual benefit of dabigatran etexilate might be reconsidered for patients with prosthetic valves, who require life-long anticoagulation and in whom S. aureus IE is associated with high mortality.

Streptococcus tigurinus is highly virulent in a rat model of experimental endocarditis.

Streptococcus tigurinus is responsible for systemic infections in humans including infective endocarditis. We investigated whether the invasive trait of S. tigurinus in humans correlated with an increased ability to induce IE in rats. Rats with catheter-induced aortic vegetations were inoculated with 10⁴ CFU/ml of either of four S. tigurinus strains AZ_3a(T), AZ_4a, AZ_8 and AZ_14, isolated from patients with infective endocarditis or with the well known IE pathogen Streptococcus gordonii (Challis). Aortic infection was assessed after 24 h. S. tigurinus AZ_3a(T), AZ_4a and AZ_14 produced endocarditis in ≥80% of rats whereas S. gordonii produced endocarditis in only 33% of animals (P<0.05). S. tigurinus AZ_8 caused vegetation infection in 56% of the animals. The capacity of S. tigurinus to induce aortic infection was not related to their ability to bind extracellular matrix proteins (fibrinogen, fibronectin or collagen) or to trigger platelet aggregation. However, all S. tigurinus isolates showed an enhanced resistance to phagocytosis by macrophages and two of them had an increased ability to enter endothelial cells, key attributes of invasive streptococcal species.

Shear-Resistant Binding to von Willebrand Factor Allows Staphylococcus lugdunensis to Adhere to the Cardiac Valves and Initiate Endocarditis

BACKGROUND Staphylococcus lugdunensis is an emerging cause of endocarditis. To cause endovascular infections, S. lugdunensis requires mechanisms to overcome shear stress. We investigated whether platelets and von Willebrand factor (VWF) mediate bacterial adhesion to the vessel wall and the cardiac valves under flow. METHODS S. lugdunensis binding to VWF, collagen, and endothelial cells was studied in a parallel flow chamber in the absence and presence of platelets. In vivo adhesion of S. lugdunensis was evaluated in a mouse microvasculature perfusion model and a new mouse model of endocarditis. RESULTS Contrary to other coagulase-negative staphylococci, S. lugdunensis bound to VWF under flow, thus enabling its adhesion to endothelial cells and to the subendothelial matrix. In inflamed vessels of the mesenteric circulation, VWF recruited S. lugdunensis to the vessel wall. In a novel endocarditis mouse model, local inflammation and the resulting release of VWF enabled S. lugdunensis to bind and colonize the heart valves. CONCLUSIONS S. lugdunensis binds directly to VWF, which proved to be vital for withstanding shear forces and for its adhesion to the vessel wall and cardiac valves. This mechanism explains why S. lugdunensis causes more-aggressive infections, including endocarditis, compared with other coagulase-negative staphylococci.

Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall.

Essentials Staphylococcus aureus (S. aureus) binds to endothelium via von Willebrand factor (VWF). Secreted VWF‐binding protein (vWbp) mediates S. aureus adhesion to VWF under shear stress. vWbp interacts with VWF and the Sortase A‐dependent surface protein Clumping factor A (ClfA). VWF‐vWbp‐ClfA anchor S. aureus to vascular endothelium under shear stress.

Failure of Vancomycin Continuous Infusion against Experimental Endocarditis Due to Vancomycin-Intermediate Staphylococcus aureus

ABSTRACT Continuous infusion of vancomycin was evaluated against experimental endocarditis due to heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and VISA. Animals were infected with hVISA PC1 (vancomycin MIC, 2 mg/liter) or VISA PC3 (vancomycin MIC, 8 mg/liter) and treated for 5 days with constant serum levels of 20 or 40 mg/liter. Vancomycin continuous infusion was unsuccessful, as 20 mg/liter was barely active against PC1 (6 of 13 sterile vegetations) and 40 mg/liter failed against PC3 (2 of 9 sterile vegetations).

In Vivo Synergism of Ceftobiprole and Vancomycin against Experimental Endocarditis Due to Vancomycin-Intermediate Staphylococcus aureus

ABSTRACT The efficacy of ceftobiprole combined with vancomycin was tested against two vancomycin-intermediate Staphylococcus aureus (VISA) strains, PC3 and Mu50, in rats with experimental endocarditis. Animals with infected aortic vegetations were treated for 3 days with doses simulating the kinetics after intravenous administration in humans of (i) the standard dose of ceftobiprole of 500 mg every 12 h (b.i.d.) (SD-ceftobiprole), (ii) a low dose of ceftobiprole of 250 mg b.i.d. (LD-ceftobiprole), (iii) a very low dose of ceftobiprole of 125 mg b.i.d. (VLD-ceftobiprole), (iv) SD-vancomycin of 1 g b.i.d., or (v) LD- or VLD-ceftobiprole combined with SD-vancomycin. Low dosages of ceftobiprole were purposely used to highlight positive drug interactions. Treatment with SD-ceftobiprole sterilized 12 of 14 (86%) and 10 of 13 (77%) vegetations infected with PC3 and Mu50, respectively (P < 0.001 versus controls). In comparison, LD-ceftobiprole sterilized 10 of 11 (91%) vegetations infected with PC3 (P < 0.01 versus controls) but only 3 of 12 (25%) vegetations infected with Mu50 (P > 0.05 versus controls). VLD-ceftobiprole and SD-vancomycin alone were ineffective against both strains (≤8% sterile vegetations). In contrast, the combination of VLD-ceftobiprole and SD-vancomycin sterilized 7 of 9 (78%) and 6 of 14 (43%) vegetations infected with PC3 and Mu50, respectively, and the combination of LD-ceftobiprole and SD-vancomycin sterilized 5 of 6 (83%) vegetations infected with Mu50 (P < 0.05 versus controls and monotherapy). Thus, ceftobiprole monotherapy simulating standard therapeutic doses was active against VISA experimental endocarditis. Moreover, subtherapeutic LD- and VLD-ceftobiprole synergized with ineffective vancomycin to restore efficacy. Hence, combining ceftobiprole with vancomycin broadens the therapeutic margin of these two compounds against VISA infections.

Bacterial adherence to graft tissues in static and flow conditions

Background Various conduits and stent‐mounted valves are used as pulmonary valve graft tissues for right ventricular outflow tract reconstruction with good hemodynamic results. Valve replacement carries an increased risk of infective endocarditis (IE). Recent observations have increased awareness of the risk of IE after transcatheter implantation of a stent‐mounted bovine jugular vein valve. This study focused on the susceptibility of graft tissue surfaces to bacterial adherence as a potential risk factor for subsequent IE. Methods Adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus sanguinis to bovine pericardium (BP) patch, bovine jugular vein (BJV), and cryopreserved homograft (CH) tissues was quantified under static and shear stress conditions. Microscopic analysis and histology were performed to evaluate bacterial adhesion to matrix components. Results In general, similar bacteria numbers were recovered from CH and BJV tissue surfaces for all strains, especially in flow conditions. Static bacterial adhesion to the CH wall was lower for S sanguinis adhesion (P < .05 vs BP patch). Adhesion to the BJV wall, CH wall, and leaflet was decreased for S epidermidis in static conditions (P < .05 vs BP patch). Bacterial adhesion under shear stress indicated similar bacterial adhesion to all tissues, except for lower adhesion to the BJV wall after S sanguinis incubation. Microscopic analysis showed the importance of matrix component exposure for bacterial adherence to CH. Conclusions Our data provide evidence that the surface composition of BJV and CH tissues themselves, bacterial surface proteins, and shear forces per se are not the prime determinants of bacterial adherence.

Vaccination against Staphylococcus aureus experimental endocarditis using recombinant Lactococcus lactis expressing ClfA or FnbpA.

Staphylococcus aureus is a major cause of serious infections in humans and animals and a vaccine is becoming a necessity. Lactococcus lactis is a non-pathogenic bacterium that can be used as a vector for the delivery of antigens. We investigated the ability of non-living L. lactis heterologously expressing S. aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnbpA), alone or together, to elicit an immune response in rats and protect them from S. aureus experimental infective endocarditis (IE). L. lactis ClfA was used for immunization against S. aureus Newman (expressing ClfA but not FnbpA), while L. lactis ClfA, L. lactis FnbpA, as well as L. lactis ClfA/FnbpA, were used against S. aureus P8 (expressing ClfA and FnbpA). Vaccination of rats with L. lactis ClfA elicited antibodies that inhibited binding of S. aureus Newman to fibrinogen, triggered the production of IL-17A and conferred protection to 13/19 (68%) of the animals from IE (P<0.05). Immunization with L. lactis ClfA, L. lactis FnbpA or L. lactis ClfA/FnbpA also produced antibodies against the target proteins, but these did not prevent binding of S. aureus P8 to fibrinogen or fibronectin and did not protect animals against S. aureus P8 IE. Moreover, immunization with constructs containing FnbpA did not increase IL-17A production. These results indicate that L. lactis is a valuable antigen delivery system able to elicit efficient humoral and cellular responses. However, the most appropriate antigens affording protection against S. aureus IE are yet to be elucidated.