Pathogenic conversion of coagulase-negative staphylococci

Abstract

Humans and animals are colonized by members of the genus Staphylococcus, however only some of these species evolved to cause invasive disease. The genetic basis for conversion of commensal staphylococci into pathogens is not known. We hypothesized that Staphylococcus aureus genes for coagulation and agglutination in vertebrate blood (coa, vwb and clfA) may support pathogenic conversion. Expression of coa and vwb in Staphylococcus epidermidis or Staphylococcus simulans supported a coagulase-positive phenotype but not the ability to cause disease in a mouse model of bloodstream infection. However, the simultaneous expression of coa, vwb and clfA in coagulase-negative staphylococci enabled bacterial agglutination in plasma and enhanced survival of S. simulans in human whole blood. Agglutination of S. simulans in the bloodstream of infected mice upon expression of coa, vwb and clfA provided also a mean for dissemination and replication in distal organs. Thus, the acquisition of genes for bacterial agglutination with fibrin appear sufficient for the conversion of commensal staphylococci into invasive pathogens.

Keywords: Abscess; Agglutination; Clumping; Coagulation; Staphylococcus; Virulence.

Fig. 1

Production of Coa, vWbp and ClfA by S. epidermidis and S. simulans. (A) Diagram illustrating plasmid inserts carrying the coa, vwb or clfA genes from S. aureus Newman. (B) Culture supernatants were examined by immunoblotting with antibodies specific for Coa (α-Coa) and vWbp (α-vWbp) while whole culture lysates were used to examine ClfA production using antibodies specific for ClfA (α-ClfA). Cells cultures were normalized A₆₀₀ 4 and S. aureus samples were diluted 10 times for Coa and vWbp or concentrated 5 times for ClfA analyses as compared to S. epidermidis and S. simulans extracts. Numbers to the left of blots indicate the position of molecular markers in kDa.

Fig. 2

Expression of coa and vwb promotes clotting by S. epidermidis and S. simulans. Anti-coagulated mouse, rabbit or human plasma was incubated with bacteria for up to 24 hours at 25°C. Tubes were tilted to assess coagulation. Data are representative of three independent experiments.

Fig. 3

Expression of clfA promotes clumping by S. epidermidis and S. simulans. Sedimentation of bacteria was monitored immediately after mixing cultures with soluble fibrinogen and inverting the tubes. Data are representative of three independent experiments.

Fig. 4

Staphylococcal agglutination in citrate-treated plasma. (A) Agglutination in human plasma of Syto-9 stained bacteria. Scale bar, 50 μm. (B) Box and whisker plot representation of agglutination areas with anti-coagulated human, mouse and rabbit plasma. Statistical significance was determined using ANOVA (****, P < 0.0001).

Fig. 5

Staphylococcal survival in human blood. Mid-log phase bacteria (1.25 × 10⁷ CFU) were inoculated into freshly drawn human blood anti-coagulated with desirudin. At 0 min and 60 min, saponin- and streptokinase-containing buffer was added to lyse host cells and release agglutinated bacteria. Bacteria were counted by plating serial dilutions on solid medium and reported as CFU/ml. Data are from three independent experiments; error bars indicate standard deviation. Statistical significance was performed by two-way ANOVA analysis, (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

Fig. 6

Pathogenic conversion of S. simulans with S. aureus coa, vwb and clfA. Mice were injected in the peri-orbital venous plexus with 5×10⁷ of S. simulans carrying either vector, pcoa-vwb, pclfA or pcoa-vwb-clfA, and euthanized 5 days following challenge. (A) Following necropsy, renal tissues were assessed for bacterial load. (B) Lesions observed in panels C through F were enumerated using 20–25 slides per infected group. The data in panels A and B represent the cumulative result of three independent experiments and were analyzed using the one-way ANOVA on ranks (Kruskal-Wallis test) with Dunn's post-hoc test (***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant). (C-F) Microscopy images of hematoxylin-eosin-stained, thin-sectioned renal tissue. Whole kidney sections are shown and areas boxed in black are shown as a 20× enlarged images. Images and insets in panels C and D did not reveal pathological features in renal tissues. In panels E and F, arrows point to replicating bacteria and immune cell infiltrates.