SATA-8505 Induces Interferon Gamma in Primary Human Keratinocytes.

<p>Human peripheral mononuclear cells (PBMC) were cultured in triplicate at 2⁶/mL with SATA-8505 at 1 PFU/mL to 10⁸ PFU/mL at ten-fold increments or diluent derived from the supernatant of an overnight culture of SA that had been pelleted and filter sterilized in a manner similar to the phage-containing media. At 72 hours supernatants were harvested and analyzed for IL-1ß (a), IL-6 (b), IL-17A (c), and IFN gamma (d). Human keratinocytes from primary foreskins (foreskin keratinocytes; FSKC) or the HaCaT cell line were cultured to confluence on 6-well plates and incubated in triplicate with SATA-8505 at 10⁴ PFU/mL to 10⁸ PFU/mL at ten-fold increments or TSB diluent. At 24 hours supernatants were harvested and analyzed for IL-1ß (e), IL-6 (f), and IFN gamma (g). Phage for all experiments was diluted in TSB from overnight culture of USA300 that was centrifuged at 5000rpm for 12 minutes and filter-sterilized through a 0.44 micrometer filter. Data shown are representative of 3 independent experiments using 3 different healthy volunteers (a-d) or a pool of 5 or more foreskin samples (e-g) and displayed as mean <u>+</u> s.e.m. * = p <0.05.</p

SATA-8505 Does Not Impact USA300 Growth in Human Blood.

<p>(a) Hourly quantification of starting culture of 10⁶ CFU USA300 in three parts TSB and one part whole blood from healthy donor or a patient with CGD, grown with or without 10⁸ PFU SATA-8505 in duplicate. (b) Regrowth of surviving colonies from whole blood culture run in triplicate. One colony of <i>S</i>. <i>aureus</i> previously grown in 3:1 TSB:Whole blood without phage was subsequently grown in TSB with SATA-8505 (TSB/Phage) or without phage (TSB/TSB); one colony of the surviving <i>S</i>. <i>aureus</i> previously grown in the presence of SATA-8505 was subsequently grown in TSB with SATA-8505 (Phage/Phage) or without phage (Phage/TSB). (c) Average quantification of starting culture of 10¹⁰ CFU of USA300 grown in either 3:1 TSB:Whole blood or 3:1 TSB:HBSS, with or without 10⁷ PFU SATA-8505. (d) Average quantification of starting culture of 10⁷ CFU of USA300 grown in three parts TSB with either one part whole blood, HBSS, serum, or peripheral mononuclear cells (PBMC) in equivalent volume HBSS. Data shown are representative of 2–3 independent experiments using 3 or more different healthy volunteers and 2 patients with CGD. Data is displayed as mean + s.e.m. ** = p<0.01.</p

SATA 8505 Effectively Kills USA 300 and Reduces its Viability in vitro.

<p>(a) Average colony forming unit (CFU) counts for USA300 cultured with SATA-8505 for up to four hours at ratios of <i>S</i>. <i>aureus</i>:Phage of 1:1, 1:10, or 1:100 (MOI 1, 10, 100 respectively). (b) Images of surviving colony morphology of USA300 grown in TSB after exposure to BHI (diluent) or SATA-8505. (c) Regrowth of surviving colonies pictured in panel b, <i>S</i>. <i>aureus</i> grown in TSB after prior exposure to BHI (diluent) or SATA-8505 run in triplicate culture. Data shown are representative of 3 or more independent experiments and displayed as mean <u>+</u> s.e.m. **** = p<0.0001.</p

SATA-8505 Improves MRSA Skin Infection at Low MOI.

<p>Mice were injected intraperitoneally (I.P.) with 10⁷ plaque-forming units (PFU) of SATA-8505 immediately prior to subcutaneous injections of 10⁷ CFU of USA300. (a) Lesion size progression over following six days. On day 6, skin biopsies were homogenized for culture, total bacterial (b) and phage burden (c) were calculated for individual mice. (d) mRNA transcript levels for IL-1ß, IL-6, and IL-17A in individual CGD mice relative to wild type controls, standardized to GAPDH. (e-g) mRNA transcript levels for IL-1ß, IL-6, and IL-17A in CGD and wild type mice with and without SATA-8505 treatment relative to wild type controls injected with diluent, standardized to GAPDH. Data shown are representative of 2–3 independent experiments using 5 or more mice per group, and displayed as mean + s.e.m. Differences were calculated by ANOVA with Bonferroni correction and depict differences from diluent treated wild type unless otherwise noted. ns = not significant, * = p <0.05, ** = p<0.01.</p

SATA-8505 Fails to Improve MRSA Skin Infection at High MOI.

<p>Lesion size (a), CFU (b), PFU (c), and transcript data (d-f) for mice injected with 10⁹ plaque-forming units (PFU) of SATA-8505 immediately prior to subcutaneous injections of 10⁷ CFU of USA300 processed in an identical manner as MOI of 1 experiments. Data shown are representative of 2–3 independent experiments using 5 or more mice per group, and displayed as mean + s.e.m. Differences were calculated by ANOVA with Bonferroni correction and depict differences from diluent treated wild type unless otherwise noted. ns = not significant, * = p <0.05, ** = p<0.01, *** = p<0.001.</p

SATA-8505 Exhibits Commercial Viability but Significant Strain Limitations.

<p>(a) SATA-8505 quantified over up to sixty days after storage at either room temperature (RT), 4 degrees Celsius (4^°C), or frozen at -80^°C. (b) Average hourly quantification of 10⁸–10⁹ CFU of USA100 <i>S</i>. <i>aureus</i> or a vancomycin resistant variant of USA100 (VRSA) cultured with up to 10¹¹ PFU of SATA-8505. Data shown are a representative of 2 independent experiments.</p