Relative inability of <i>S</i>. <i>aureus</i> to bind B cells is conserved among diverse strains and influenced by bacterial viability.

<p><b>A)</b> Association of B cells with FITC-labeled <i>S</i>. <i>aureus</i> strains (USA100, USA200, and USA300) or <i>S</i>. <i>epidermidis</i> strains (1457, 12228, and RP62A) in heparinized human blood was determined by flow cytometry (60 min time point). ~2.5–5 × 10⁵ CFU/mL of bacteria were used in these assays. <b>B)</b> Association of B cells with untreated or UV-irradiated USA300 or <i>S</i>. <i>epidermidis</i> strain 1457 (1 × 10⁷ CFU/mL for each) in heparinized human blood was determined by flow cytometry (40 min time point). Data in panels A and B are the mean ± SEM of 5 separate experiments using different blood donors. For panel A, ^#, ^, *<i>P</i>≤0.002 versus 1457, 12228, or RP62A as determined by repeated-measures one-way ANOVA and Tukey’s post-test. For panel B, **<i>P</i><0.01 as determined by paired two-tailed Student’s t test. NS, not significant.</p

Association of <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i> with PMNs, monocytes, B cells, and T cells in human blood.

<p><b>A</b>) Representative flow cytometry dot plots of CD3⁺ lymphocytes (T cells), CD19⁺ lymphocytes (B cells), CD14⁺ monocytes, and CD66^High granulocytes (PMNs) 40 min after combining heparinized human blood with FITC-labeled <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i>. <b>B-E</b>) Flow cytometry analyses of leukocyte subsets associated with FITC-labeled <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i>. <b>F & G</b>) Relative distribution of CD66^High granulocytes, CD14⁺ monocytes, CD19⁺ lymphocytes, and CD3⁺ lymphocytes associated with <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i> at 40 min. Data are from experiments presented in panels B-E. Data in all panels represent 3 experiments with different blood donors using 5 × 10⁵ CFU/mL bacteria. *<i>P</i><0.05 and **<i>P</i><0.01 versus <i>S</i>. <i>aureus</i> as determined with a paired two-tailed Student’s t test.</p

Association of human B cells with <i>S</i>. <i>epidermidis</i> is mediated primarily by complement.

<p><b>A)</b> FITC-labeled <i>S</i>. <i>epidermidis</i> (strain 1457) was opsonized with NHS, heat-treated NHS (HT Tx), or DPBS (unopsonized control) and binding of B cells to <i>S</i>. <i>epidermidis</i> was assessed by flow cytometry. <b>B)</b> FITC-labeled <i>S</i>. <i>epidermidis</i> was opsonized with NHS, NHS treated with cobra venom factor (+CVF), NHS treated with FUT-175 (+FUT-175), HT Tx, or DPBS and binding of B cells to <i>S</i>. <i>epidermidis</i> was assessed by flow cytometry (60 min time point). Data in panels <b>A</b> and <b>B</b> are the mean ± SEM of 5 separate experiments using different blood donors. <b>C)</b> FITC-labeled <i>S</i>. <i>epidermidis</i> was combined with 5 μg anti-human CD35 (+αCR1), CD21 (+αCR2), CD11b (+αCR3), CD11c (+αCR4), or isotype IgG₂ control (isotype) mAbs and binding to B cells was determined using flow cytometry (30 min time point). <b>D)</b> FITC-labeled <i>S</i>. <i>epidermidis</i> was combined with 4 μg of soluble recombinant CD35 (+rCR1), CD21 (+rCR2), CD11b (+rCR3), CD11c (+rCR4), or DPBS and binding to B cells was determined using flow cytometry (60 min time point). Data in panels C and D are the mean ± SEM of at least 3 separate experiments using different blood donors. Experiments in panels A-D were performed using 1 × 10⁶ purified human peripheral blood mononuclear cells (PBMCs) and 1 × 10⁶ CFUs <i>S</i>. <i>epidermidis</i>. B cells were identified as the CD19⁺ leukocyte population of PBMCs. **<i>P</i><0.01, ***<i>P</i><0.001, and ****<i>P</i><0.0001 versus DPBS controls for panels A and D, NHS for panel B, or isotype IgG₂ for panel C. Statistics were determined by using a repeated-measures one-way ANOVA and Dunnett’s post-test.</p

Comparison of the binding of <i>S</i>. <i>aureus</i>, <i>S</i>. <i>epidermidis</i> and zymosan with human B cells.

<p><b>A)</b> Flow cytometry analysis of B cells associated with <i>S</i>. <i>aureus</i> strain USA300 or <i>S</i>. <i>epidermidis</i> strain 1457 in heparinized human blood 40 min after the addition of 5 × 10⁵, 1 × 10⁶, or 1 × 10⁷ CFU/mL of staphylococci. <b>B)</b> Percentage of B cells with bound bacteria or zymosan 40 min after combining heparinized human blood with 8 × 10⁶ CFU/mL of FITC-labeled USA300 or <i>S</i>. <i>epidermidis</i> strain 1457, or the indicated numbers of FITC-labeled zymosan particles. Data in panels A and B are the mean ± SEM of at least 4 separate experiments using different blood donors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, and ****<i>P</i><0.0001 versus USA300 as determined by paired two-tailed Student’s t test for data in panel A or by repeated-measures one-way ANOVA and Dunnett’s post-test for data in panel B.</p

Immunofluorescence microscopy analysis of purified B cells and monocytes associated with <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i>.

<p><b>A</b>) Representative images (100×) of B cells or monocytes purified from heparinized human blood 80 min after addition of 1 × 10⁶ CFU/mL <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i>. Extracellular bacteria are red and internalized bacteria are green (appear turquoise in the context of blue nuclei). <b>B</b>) Quantitation of the association and internalization of <i>S</i>. <i>aureus</i> or <i>S</i>. <i>epidermidis</i> by B cells or monocytes for assays described in panel A. Data in panel B are the mean ± SEM of at least 5 separate experiments. *<i>P</i><0.05 for comparison of <i>S</i>. <i>aureus</i> with <i>S</i>. <i>epidermidis</i> as determined by paired two-tailed Student’s t test.</p