Figure 1.
MSSA CNS catheter-associated biofilm infection persists longer than a parenchymal brain abscess.
The infected tissue was removed, homogenized and cultured to enumerate bacterial burdens in the tissue containing the brain abscess or surrounding the infected catheter. Catheters were also removed, rinsed and sonicated for quantification of viable bacteria. * = p<0.05 (n = 12–18 mice/group/time point).
Figure 2.
CNS catheter-associated biofilm infection is associated with attenuated inflammation compared to parenchymal abscesses.
The tissues encompassing the brain abscess and the tissues surrounding the infected catheters were homogenized and the resulting supernatants analyzed for levels of the pro-inflammatory mediators CXCL1 (A, B) and IL-17 (C, D). Raw cytokine/chemokine levels are reported (A, C) as well as correction for disparate bacterial burdens (B, D) * = p<0.05 (n = 12–18 mice/group/time point).
Figure 3.
sarA is critical for catheter-associated biofilm formation in a CNS catheter infection model.
Infected catheters were removed, rinsed and sonicated for quantification of viable bacteria associated with wild type ACH1719 (WT) or ACH1719ΔsarA (ΔsarA) S. aureus. At all time points, bacterial burdens in brain parenchyma were lower than the corresponding catheter cultures (See Figure S2). * = p<0.05 WT catheter vs ΔsarA catheter; (n = 11–21 mice/group/time point).
Figure 4.
sarA dictates the extent of proinflammatory mediator expression during CNS catheter-associated infection.
Catheter-associated infections were generated with either wild type ACH1719 (WT) or ACH1719ΔsarA (ΔsarA). The tissue surrounding the infected catheters was homogenized and the resulting supernatant analyzed for levels of the pro-inflammatory mediators CXCL1 (A, B) and IL-1β (C, D). Results are presented as raw data (A and C) and after adjustment for divergent bacterial burdens (B and D). * = p<0.05 (n = 11–21 mice/group/time point).
Figure 5.
sarA influences peripheral immune cell influx during CNS catheter-associated infection.
Catheter associated cells were recovered from mice at days 3, 7 and 14-infection, whereupon pooled tissue from 4–5 mice per group (wild type ACH1719– WT vs ACH1719ΔsarA – ΔsarA) was analyzed for neutrophil (A), macrophage (B) and T cell (C) infiltrates, with three independent replicates. Data was corrected for bacterial burdens at each time point. See Figure S2 for representative histograms.
Figure 6.
α-toxin levels are reduced during CNS catheter-associated infection with a sarA mutant.
Alpha-toxin levels were measured in brain homogenates of animals infected with the ACH1719 sarA mutant (ΔsarA) or wild type ACH1719 strain (WT) at the indicated time points post-infection using a custom-designed ELISA. * = p<0.05 (n = 13–15 mice/group/time point).
Figure 7.
sarA and extracellular proteases significantly affect biofilm formation in a CNS catheter infection model.
Infected catheters were removed, rinsed and sonicated for quantification of viable bacteria associated with wild type USA300 LAC (WT catheter), USA300 LACΔsarA (SarA-deficient catheter), extracellular protease deficient USA300 LAC (Protease-deficient catheter), or SarA and extracellular protease deficient (SarA-Protease-deficient catheter) S. aureus. At all time points, bacterial burdens in brain parenchyma were 3–5 log lower than the corresponding catheter cultures (See Figure S4). a = p<0.05 WT vs SarA-deficient catheter; b = p<0.05 WT vs Protease-deficient catheter; c = p<0.05 WT vs SarA-Protease-deficient catheter; d = p<0.05 SarA-deficient vs Protease-deficient catheter; e = SarA-deficient vs SarA-Protease-deficient catheter; f = SarA-Protease-deficient vs Protease-deficient catheter (n = 13–15 mice/group/time point).
Figure 8.
Inflammatory mediators are elevated in response to infection with sarA and sarA-protease mutants independently of protease activity.
Catheter-associated infections were generated with wild type USA300 LAC (WT catheter), USA300 LACΔsarA (SarA-deficient catheter), extracellular protease deficient USA300 LAC (Protease-deficient catheter), or SarA and extracellular protease deficient (SarA-Protease-deficient catheter) S. aureus. The tissue surrounding the infected catheters was homogenized and the resulting supernatant analyzed for levels of the pro-inflammatory mediators CXCL1 (A, B) and IL-1β (C, D). Results are presented as raw data (A and C) and after adjustment for divergent bacterial burdens (B and D). * = p<0.05 (n = 13–15 mice/group/time point).