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The mechanisms or host factors involved in septic thrombus or vegetation formation in Staphylococcus aureus-induced infective endocarditis (IE) are unclear. Using an experimental endocarditis rat model, here we demonstrated that S. aureus HG001-induced vegetation was composed of bacterial floes encased in aggregated platelets and surrounded by neutrophil extracellular traps (NETs). In vitro data demonstrated that platelets contribute to both biofilm and NET formation. Prophylactic administration of DNase I significantly reduced the size of vegetation induced by methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains, even though MRSA and MSSA isolates express different biofilm phenotypes and NET-induction abilities in the presence of platelets. Moreover, delivery of both DNase I and daptomycin prophylactically and therapeutically produced synergistic effects by reducing vegetation size and bacterial numbers on damaged valve tissues in MRSA-induced IE. Together, these data suggest that NETs contribute to vegetation formation in S. aureus endocarditis and DNase I has the potential to control S. aureus-induced IE in the clinic.

Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (Δ atlA ) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an Δ atlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the Δ atlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in Δ atlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans , which contributes to biofilm formation in infective endocarditis.

Infective endocarditis is a typical biofilm-associated infectious disease frequently caused by commensal streptococci, but the contribution of host factors in biofilm formation is unclear. We found that platelets are essential for in vitro biofilm formation by Streptococcus mutans or Streptococcus gordonii grown in human plasma. The biofilms were composed of bacterial floes embedded with platelet aggregates in layers, and a similar architecture was also detected in situ on the injured valves of a rat model of experimental endocarditis. Similar to planktonic cells, the streptococci in biofilms were also able to induce platelet aggregation, which facilitates multilayer biofilm formation. Entrapping of platelets directly enhances the resistance of streptococcal biofilms to clindamycin. Prophylactic antibiotics or aspirin can reduce but not prevent or abolish biofilm formation on injured heart valves. Therefore, the platelet is a host factor for commensal streptococci in the circulation to consolidate biofilm formation and protect bacteria against antibiotics.

Background— Endocarditis-inducing streptococci form multilayered biofilms in complex with aggregated platelets on injured heart valves, but the host factors that interconnect and entrap these bacteria-platelet aggregates to promote vegetation formation were unclear. Methods and Results— In a Streptococcus mutans endocarditis rat model, we identified layers of neutrophil extracellular traps interconnecting and entrapping bacteria-platelet aggregates inside vegetation that could be reduced significantly in size along with diminished colonizing bacteria by prophylaxis with intravascular DNase I alone. The combination of activated platelets and specific immunoglobulin G–adsorbed bacteria are required to induce the formation of neutrophil extracellular traps through multiple activation pathways. Bacteria play key roles in coordinating the signaling through spleen tyrosine kinase, Src family kinases, phosphatidylinositol-3-kinase, and p38 mitogen-activated protein kinase pathways to upregulate the expression of P-selectin in platelets, while inducing reactive oxygen species–dependent citrullination in the arm of neutrophils. Neutrophil extracellular traps in turn serve as the scaffold to further enhance and entrap bacteria-platelet aggregate formation and expansion. Conclusions— Neutrophil extracellular traps promote and expand vegetation formation through enhancing and entrapping bacteria-platelet aggregates on the injured heart valves. (Circulation. 2015;131:571–581. DOI: 10.1161/CIRCULATIONAHA.114.011432.)

Streptococcus mutans, a commensal of the human oral cavity, can survive in the bloodstream and cause infective endocarditis (IE). However, the virulence factors associated with this manifestation of disease are not known. Here, we demonstrate that AtlA, an autolysin of S. mutans is a newly identified fibronectin (Fn) binding protein and contributes to bacterial resistance to phagocytosis and survival in the bloodstream. Interestingly, prior exposure to plasma at low concentrations was sufficient to enhance bacterial survival in the circulation. Calcium ions at physiological plasma concentrations induced maturation of AtlA from the 104-90 kDa isoform resulting in increased Fn binding and resistance to phagocytosis. An isogenic mutant strain defective in AtlA expression exhibited reduced survival and virulence when tested in a rat model of IE compared with the wild-type and complemented strains. The data presented suggest that plasma components utilized by S. mutans enhanced survival in the circulation and AtlA is a virulence factor associated with infective endocarditis.