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1.
Eur Heart J ; 40(39): 3248-3259, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-30945735

RESUMO

AIMS: The pathogenesis of endocarditis is not well understood resulting in unsuccessful attempts at prevention. Clinical observations suggest that Staphylococcus aureus infects either damaged or inflamed heart valves. Using a newly developed endocarditis mouse model, we therefore studied the initial adhesion of S. aureus in both risk states. METHODS AND RESULTS: Using 3D confocal microscopy, we examined the adhesion of fluorescent S. aureus to murine aortic valves. To mimic different risk states we either damaged the valves with a surgically placed catheter or simulated valve inflammation by local endothelium activation. We used von Willebrand factor (VWF) gene-deficient mice, induced platelet and fibrinogen depletion and used several S. aureus mutant strains to investigate the contribution of both host and bacterial factors in early bacterial adhesion. Both cardiac valve damage and inflammation predisposed to endocarditis, but by distinct mechanisms. Following valve damage, S. aureus adhered directly to VWF and fibrin, deposited on the damaged valve. This was mediated by Sortase A-dependent adhesins such as VWF-binding protein and Clumping factor A. Platelets did not contribute. In contrast, upon cardiac valve inflammation, widespread endothelial activation led to endothelial cell-bound VWF release. This recruited large amounts of platelets, capturing S. aureus to the valve surface. Here, neither fibrinogen, nor Sortase A were essential. CONCLUSION: Cardiac valve damage and inflammation predispose to S. aureus endocarditis via distinct mechanisms. These findings may have important implications for the development of new preventive strategies, as some interventions might be effective in one risk state, but not in the other.


Assuntos
Valva Aórtica/microbiologia , Aderência Bacteriana , Endocardite Bacteriana/microbiologia , Inflamação/complicações , Infecções Estafilocócicas/complicações , Staphylococcus aureus/fisiologia , Animais , Valva Aórtica/lesões , Plaquetas , Coagulase/metabolismo , Modelos Animais de Doenças , Endocardite Bacteriana/metabolismo , Endotélio/metabolismo , Feminino , Fibrina/metabolismo , Inflamação/metabolismo , Masculino , Camundongos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
2.
Pediatr Infect Dis J ; 38(5): 453-458, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30346369

RESUMO

BACKGROUND: Infective endocarditis (IE) remains a diagnostic and therapeutic challenge associated with high morbidity and mortality. We evaluated the microbial profile and clinical manifestation of IE in children. METHODS: A retrospective study examining pediatric IE cases treated between 2000 and 2017 at the Department of Pediatric Cardiology, KU Leuven, was conducted. Clinical presentation, treatment, complications, outcome of IE, underlying microorganisms and congenital heart defects were reviewed. RESULTS: Fifty-three patients were diagnosed with IE. Overall, 19 patients (36%) required cardiac surgery. Seven patients (13%) died. Eighty-seven percent of patients had an underlying congenital cardiac defect. Eighteen (34%) children presented with prosthetic graft IE. A causative organism was found in 49 (92%) cases: viridans group streptococci were identified in 17 (32%), Staphylococcus aureus in 13 (25%) and coagulase-negative staphylococci in 11 (20%) children. Community-acquired (CA) IE increased significantly from 8 (33%) cases in 2000-2007 to 20 (74%) cases in 2008-2017 (P < 0.01). Even with viridans streptococci being significantly more prevalent in the CA group (P < 0.01), we did not observe an increase of streptococcal IE from 2008 to 2017. Seventeen (32%) patients presented with hospital-acquired IE during the first year of life with 14 (82%) children after surgery and a prevalence of coagulase-negative staphylococci (53%). CONCLUSIONS: The incidence of pediatric IE was similar over the investigated time period with a shift toward CA IE. Streptococci and staphylococci accounted for the majority of cases in both periods. Awareness of IE and its prevention is crucial in patients after implantation of prosthetic grafts.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Endocardite/microbiologia , Endocardite/patologia , Adolescente , Bactérias/classificação , Infecções Bacterianas/mortalidade , Infecções Bacterianas/terapia , Bélgica/epidemiologia , Criança , Pré-Escolar , Endocardite/mortalidade , Endocardite/terapia , Feminino , Hospitais Pediátricos , Hospitais Universitários , Humanos , Lactente , Recém-Nascido , Masculino , Prevalência , Estudos Retrospectivos , Análise de Sobrevida , Resultado do Tratamento
3.
Virulence ; 9(1): 1615-1624, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30280967

RESUMO

Staphylococcus aureus is the leading cause of infective endocarditis (IE). While the role of S. aureus cell-wall associated protein clumping factor A (ClfA) in promoting IE has been already demonstrated, that of the secreted plasma-clotting factors staphylocoagulase (Coa) and von Willebrand factor-binding protein (vWbp) has not yet been elucidated. We investigated the role of Coa and vWbp in IE initiation in rats with catheter-induced aortic vegetations, using Lactococcus lactis expressing coa, vWbp, clfA or vWbp/clfA, and S. aureus Newman Δcoa, ΔvWbp, ΔclfA or Δcoa/ΔvWbp/ΔclfA mutants. vWbp-expression increased L. lactis valve infection compared to parent and coa-expressing strains (incidence: 62%, versus 0% and 13%, respectively; P < 0.01). Likewise, expression of clfA increased L. lactis infectivity (incidence: 80%), which was not further affected by co-expression of vWbp. In symmetry, deletion of the coa or vWbp genes in S. aureus did not decrease infectivity (incidence: 68 and 64%, respectively) whereas deletion of clfA did decrease valve infection (incidence: 45%; P = 0.03 versus parent), which was not further affected by the triple deletion Δcoa/ΔvWbp/ΔclfA (incidence: 36%; P > 0.05 versus ΔclfA mutant). Coa does not support the initial colonization of IE (in L. lactis) without other key virulence factors and vWbp contributes to initiation of IE (in L. lactis) but is marginal in the present of ClfA.


Assuntos
Valva Aórtica/microbiologia , Proteínas de Bactérias/metabolismo , Coagulase/metabolismo , Endocardite Bacteriana/patologia , Staphylococcus aureus/genética , Fator de von Willebrand/metabolismo , Animais , Valva Aórtica/fisiopatologia , Proteínas de Bactérias/genética , Infecções Relacionadas a Cateter/microbiologia , Coagulase/genética , Feminino , Deleção de Genes , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Ratos , Ratos Wistar , Infecções Estafilocócicas , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética
4.
Thromb Haemost ; 118(7): 1230-1241, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29909601

RESUMO

Adhesion of Staphylococcus aureus to endothelial cells (ECs) is paramount in infective endocarditis. Bacterial proteins such as clumping factor A (ClfA) and fibronectin binding protein A (FnbpA) mediate adhesion to EC surface molecules and (sub)endothelial matrix proteins including fibrinogen (Fg), fibrin, fibronectin (Fn) and von Willebrand factor (vWF). We studied the influence of shear flow and plasma on the binding of ClfA and FnbpA (including its sub-domains A, A16+, ABC, CD) to coverslip-coated vWF, Fg/fibrin, Fn or confluent ECs, making use of Lactococcus lactis, expressing these adhesins heterologously. Global adherence profiles were similar in static and flow conditions. In the absence of plasma, L. lactis-clfA binding to Fg increased with shear forces, whereas binding to fibrin did not. The degree of adhesion of L. lactis-fnbpA to EC-bound Fn and of L. lactis-clfA to EC-bound Fg, furthermore, was similar to that of L. lactis-clfA to coated vWF domain A1, in the presence of vWF-binding protein (vWbp). Yet, in plasma, L. lactis-clfA adherence to activated EC-vWF/vWbp dropped over 10 minutes by 80% due to vWF-hydrolysis by a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 and that of L. lactis-fnbpA likewise by > 70% compared to the adhesion in absence of plasma. In contrast, plasma Fg supported high L. lactis-clfA binding to resting and activated ECs. Or, in plasma S. aureus adhesion to active endothelium occurs mainly via two complementary pathways: a rapid but short-lived vWF/vWbp pathway and a stable integrin-coupled Fg-pathway. Hence, the pharmacological inhibition of ClfA-Fg interactions may constitute a valuable additive treatment in infective endocarditis.


Assuntos
Proteína ADAMTS13/sangue , Aderência Bacteriana , Coagulase/metabolismo , Endocardite Bacteriana/microbiologia , Células Endoteliais da Veia Umbilical Humana/microbiologia , Plasma/enzimologia , Staphylococcus aureus/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Células Cultivadas , Coagulase/genética , Endocardite Bacteriana/sangue , Fibrina/metabolismo , Fibrinogênio , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Staphylococcus aureus/genética , Estresse Mecânico , Fator de von Willebrand/metabolismo
5.
J Thorac Cardiovasc Surg ; 155(1): 325-332.e4, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28712577

RESUMO

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.


Assuntos
Aderência Bacteriana/fisiologia , Bioprótese , Endocardite Bacteriana , Implante de Prótese de Valva Cardíaca/efeitos adversos , Próteses Valvulares Cardíacas , Infecções Estafilocócicas , Staphylococcus , Animais , Bioprótese/efeitos adversos , Bioprótese/microbiologia , Bovinos , Endocardite Bacteriana/etiologia , Endocardite Bacteriana/prevenção & controle , Próteses Valvulares Cardíacas/efeitos adversos , Próteses Valvulares Cardíacas/microbiologia , Implante de Prótese de Valva Cardíaca/métodos , Humanos , Veias Jugulares/transplante , Infecções Relacionadas à Prótese/etiologia , Infecções Relacionadas à Prótese/prevenção & controle , Valva Pulmonar/cirurgia , Infecções Estafilocócicas/etiologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus/classificação , Staphylococcus/fisiologia , Propriedades de Superfície , Válvulas Venosas/transplante , Obstrução do Fluxo Ventricular Externo/cirurgia
6.
J Infect Dis ; 213(7): 1148-56, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26743845

RESUMO

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.


Assuntos
Aderência Bacteriana/fisiologia , Endocardite Bacteriana/microbiologia , Valvas Cardíacas/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus lugdunensis/fisiologia , Fator de von Willebrand/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Resistência ao Cisalhamento , Fator de von Willebrand/genética
7.
J Vis Exp ; (100): e52862, 2015 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-26131651

RESUMO

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood. To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.


Assuntos
Aderência Bacteriana/fisiologia , Técnicas Bacteriológicas/métodos , Vasos Sanguíneos/microbiologia , Animais , Técnicas Bacteriológicas/instrumentação , Técnicas In Vitro , Camundongos , Microscopia de Fluorescência , Staphylococcus aureus/fisiologia
8.
BMC Microbiol ; 14: 310, 2014 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-25515118

RESUMO

BACKGROUND: Staphylococcus aureus (S. aureus) is a frequent cause of skin and soft tissue infections. A unique feature of S. aureus is the combined presence of coagulases that trigger fibrin formation and of the plasminogen activator staphylokinase (SAK). Whereas the importance of fibrin generation for S. aureus virulence has been established, the role of SAK remains unclear. We studied the role of plasminogen activation by SAK in a skin infection model in mice and evaluated the impact of alpha-2-antiplasmin (α2AP) deficiency on the spreading and proteolytic activity of S. aureus skin infections. The species-selectivity of SAK was overcome by adenoviral expression of human plasminogen. Bacterial spread and density was assessed non-invasively by imaging the bioluminescence of S. aureus Xen36. RESULTS: SAK-mediated plasmin activity increased the local invasiveness of S. aureus, leading to larger lesions with skin disruption as well as decreased bacterial clearance by the host. Even though fibrin and bacterial surfaces protected SAK-mediated plasmin activity from inhibition by α2AP, the deficiency of α2AP resulted in increased bacterial spreading. SAK-mediated plasmin also induced secondary activation of gelatinases, shown both in vitro and in lesions from the in vivo model. CONCLUSION: SAK contributes to the phenotype of S. aureus skin infections by enhancing bacterial spreading as a result of fibrinolytic and proteolytic activation.


Assuntos
Fibrinolisina/metabolismo , Interações Hospedeiro-Patógeno , Metaloendopeptidases/metabolismo , Plasminogênio/metabolismo , Pele/microbiologia , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Animais , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/patologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus/fisiologia
9.
Blood ; 124(10): 1669-76, 2014 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-24951431

RESUMO

Adhesion of Staphylococcus aureus to blood vessels under shear stress requires von Willebrand factor (VWF). Several bacterial factors have been proposed to interact with VWF, including VWF-binding protein (vWbp), a secreted coagulase that activates the host's prothrombin to generate fibrin. We measured the adhesion of S aureus Newman and a vWbp-deficient mutant (vwb) to VWF, collagen, and activated endothelial cells in a microparallel flow chamber. In vivo adhesion of S aureus was evaluated in the mesenteric circulation of wild-type (WT) and VWF-deficient mice. We found a shear-dependent increase in adhesion of S aureus to the (sub)endothelium that was dependent on interactions between vWbp and the A1-domain of VWF. Adhesion was further enhanced by coagulase-mediated fibrin formation that clustered bacteria and recruited platelets into bacterial microthrombi. In vivo, deficiency of vWbp or VWF as well as inhibition of coagulase activity reduced S aureus adhesion. We conclude that vWbp contributes to vascular adhesion of S aureus through 2 independent mechanisms: shear-mediated binding to VWF and activation of prothrombin to form S aureus-fibrin-platelet aggregates.


Assuntos
Aderência Bacteriana/genética , Endotélio Vascular/microbiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Staphylococcus aureus/fisiologia , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/microbiologia , Células Cultivadas , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organismos Geneticamente Modificados , Infecções Estafilocócicas/microbiologia , Estresse Mecânico , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
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