SMSB4 promotes bacteria growth in a concentration dependent manner (C) similarly to CVF (D). S. aureus Xen29 or pyoderma isolates MRSA strains (HS16, M34), MSSA strains (HS56, M5) were harvested from mid-log growth phase culture. Bacteria (1×10⁵ cfu/ml) were challenged with whole blood pre-treated with either 100 µg/ml SMSB4, positive control 10 µg/ml CVF, negative controls 100 µg/ml BSA or GVB²⁺ buffer only. S. aureus cells in PBS only without blood was also included to illustrate that the reduction in bacteria number was due to blood killing (A). Numbers of bacteria were counted as cfu/ml at various time points (A) or at 3 h (B, C, D). Bacterial recovery was calculated as a percentage of the challenge dose. Results are shown as means ± SEM from three independent experiments. The statistical significance of differences between samples was estimated using two way ANOVA with Tukey’s multiple comparison test. **, p<0.01; ***, p<0.001; ****, p<0.0001, ns, not significant (B).

https://doi.org/10.1371/journal.pntd.0002928.g001

The wells of 96-well microtiter plates were coated with 100 µl aliquots of bacterial cell suspensions containing 5×10⁶ cfu/ml of S. aureus. Wells were then incubated with 10% NHS which has been pre-treated with increasing concentrations of either SMSB4 or BSA. Antibodies were detected by ELISA using primary human specific antibodies, followed by HRP-conjugated secondary antibodies, and fluorescence was detected at 490 nm. Results are shown as means ± SEM from three independent experiments. The statistical significance of differences between BSA and SMSB4 treated samples were estimated using two way ANOVA with Sidak’s multiple comparison test. **, p<0.01; ***, p<0.001; ns, not significant (B).

https://doi.org/10.1371/journal.pntd.0002928.g004