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 10(6) 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.

Fig 1

L. lactis-induced platelet aggregation measured by light transmission aggregometry. Results are expressed as means ± standard deviations for three independent assays. (A) Lag time (i.e., the time from the addition of bacteria to the platelets to the first signs of aggregation). Lag times of 10 min indicate that there was no aggregation even when the time was extended to 40 min. (B) Maximal aggregation (i.e., the maximum extent of aggregation over 40 min). Asterisks indicate a significant difference (P < 0.05) from the results for L. lactis pIL253 (by an unpaired t test).

Fig 2

Ability of L. lactis to trigger platelet aggregation under high-shear conditions (rate, 800 s⁻¹), determined by using μ-Slide VI^0.4 flow chambers. Images were taken 3 min after platelet perfusion over immobilized bacteria (i.e., the longest lag time for aggregating strains) and are representative fields for 3 independent experiments that yielded similar results. The percentage of chamber surface coverage (platelet aggregation) is given next to each image. For the control, a suspension of PRP was perfused over a chamber without adherent bacteria. Asterisks indicate a significant difference (P < 0.001) from the result for pIL253 (by an unpaired t test).

Fig 3

Experimental endocarditis induced by bolus or continuous-infusion inoculation. Rats with catheter-induced aortic vegetations were challenged either with an i.v. bolus (1 ml in 1 min) of 10⁶ CFU or with the same total absolute numbers of L. lactis delivered as a continuous infusion of 0.0017 ml/min over 10 h. Bars indicate the percentages of infected vegetations for each inoculation method. The recombinant strains of L. lactis are given under the bars, and the factors expressed by these strains are given above the bars. Fg, fibrinogen binding; Fn, fibronectin binding; PlAgg, platelet aggregation. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant (by Fisher's exact test).

Fig 4

Levels of TNF, IL-1α, IL-1β, and IL-6 in the plasma of rats 2 h and 6 h after the initiation of inoculation with L. lactis pIL253, L. lactis expressing ClfA, or L. lactis expressing FnbpA by either bolus (left) or low-grade continuous infusion (right). Horizontal dashed lines represent background values at 0 h. Results are expressed as means ± standard errors of the means for three values per group. Asterisks indicate a significant difference (P < 0.05) from cytokine production in L. lactis pIL253 as determined by ANOVA with Tukey's multiple-comparison test.