Fig. 2.

Lysis of human PMNs after phagocytosis of USA300 is MOI dependent and requires live bacteria. a PMNs were cultured with strains LAC (USA300 CA-MRSA) and COL (early HA-MRSA) for 6 h and PMN lysis was determined by release of LDH. PMN-tobacteria ratio was 1: 1, 1: 5 and 1: 10 as indicated. b Strain LAC was cultured to early exponential phase of growth in the indicated medium or killed as indicated and incubated with human PMNs for 6 h (MOI 10 bacteria per PMN). c PMNs were cultured with wild-type (LAC) or isogenic lukS/F-PV -negative (LAC δpvl) USA300 strains for 3 or 6 h and PMN lysis was determined by release of LDH. d PMNs were cultured with strain LAC for 30 min to promote phagocytosis, after which gentamicin (LAC + gent; 5 μg/ml) or lysostaphin (LAC + lyso; 6.25 U/ml) was added to the assay to estimate potential contribution of extracellular bacteria. Neither gentamicin nor lysostaphin alone (+ gent, + lyso) caused release of LDH by human PMNs. To estimate contribution of secreted USA300 cytolytic toxins to PMN lysis, PMNs were incubated for 6 h with sterile-filtered LAC culture supernatants (LAC SN) and LDH release was determined. Statistical analyses for a, c and e were performed using a paired t test: * p < 0.001 for LAC vs. COL in a; * p < 0.05 for serum opsonized vs. unopsonized LAC in e. Statistical analysis for d was performed using a one-way ANOVA and Dunnett's post-test: * p < 0.05 vs. LAC. Results are the mean 8 standard deviation of 6 (a), 2-6 (b), 3 (c), 3 (d) or 12 (e) PMN donors. n.s. = Not significant.