Omega-3 consumption altered MRSA susceptibility.

<p>(a) Survival after infection with <i>E. coli</i> 01 K18 in pups of breeders exposed to LF or n−3 diet (n = 10–12). (b–g) <i>Staphylococcus aureus</i> (MRSA USA300) skin infection in male BALB/c mice. Lesion sizes (b), CFU (c), and mRNA expression in skin abscess tissue normalized against LF controls (dotted line) (d) (n = 5–6) in offspring mice. Lesion sizes (e), CFU (f), and mRNA expression in skin abscess tissue normalized against LF controls (dotted line) (g) (n = 10) in adult male mice. Results are representative of two (d and g) or combined from 2–3 (a–c and e–f) independent experiments and displayed as mean+s.e.m. Significance determined by t test (b–c, e–f), ANOVA with Bonferroni’s correction (d and g), or Kaplan-Meier (a). n designates mouse number per group.</p

Fatty acid content and source for the diets studied.

<p>Breakdown of dietary components in the diets studied are shown, including protein, carbohydrates (carb), fat, and % of fat that was saturated, poly-unsaturated fatty acids (PUFA), or mono-unsaturated fatty acids (MUFA). The dietary source for each fatty acid is shown. Both diets were derived from natural oils. Human recommended diet (RD) reflects the guidelines of the United States Department of Agriculture. See Methods section for further details on diet formulation.</p

Summary of study design.

<p>(a) For experiments evaluating the effects of parental diet, littermate mice were placed on either Low Fat or high omega-3 formulations one day prior to being placed in breeding cages. Breeder mice were maintained on the different diets throughout gestation and nursing. When the pups were three weeks post-partum, they were weaned to new cages. All pups were weaned onto the Low Fat control diet. Two to four weeks after weaning, the mice were evaluated in the described models. (b) For evaluation of the effects of active diet consumption, the converse experiment was performed. Pups from breeders on the Low Fat control diet were weaned into new cages and placed on either the omega-3 or Low Fat control diet. Figure modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087181#pone.0087181-Myles1" target="_blank">[13]</a>.</p

High omega-3 intake altered allergic responses.

<p>(a–d) Weaned male BALB/c pups were gavaged with peanut protein and cholera toxin weekly for 4–8 weeks before challenge. Temperature drop after intraperitoneal challenge (a), total IgE (b), and peanut-specific antibodies (c and d) (n = 5). LF, Low Fat; n−3, Omega-3 diet. Results are representative of 2 or more (b-d) or combined from two (a) independent experiments and displayed as mean+s.e.m. Significance determined by t test. n designates mouse number per group per experiment.</p

High omega-3 intake altered colonic inflammation and gut microbiome.

<p>(a) 16S ribosomal RNA genes in cecal stool samples of female mice. Each bar represents one mouse. Phyla (Bacterioidetes, Firmicutes, or other) are classified into genus or unclassified family members. Further breakdown of composition within each phylum can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087181#pone-0087181-t002" target="_blank">Table 2</a>. (b-f) Cytokine production from female excised colons stimulated with LPS for 24–72 hours (n = 4–6). (g) Cytokine production from male splenocytes stimulated with LPS for 24–72 hours (n = 4–6). Results are representative of 2–3 independent experiments in BALB/c mice and displayed as mean+s.e.m. Significance determined by t test. All experiments were repeated with similar results in both genders. Gender is indicated when representative experiments are shown; otherwise, data reflects both male and female mice with matched ratios within experiments. n designates number of mice per experiment. The results from the Low Fat group shown in (a) are a subset of what was previously reported in reference 13. The remaining data is novel.</p

n−3 pups had altered microbiomes.

<p>16S ribosomal RNA genes in cecal stool samples as percent of total yield. Pups from indicated breeder diets were weaned to LF diet either in cages with their littermates. Each column represents one mouse. The column order of mice mirrors the presentation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087181#pone-0087181-g002" target="_blank">Figure 2a</a>. LF, Low Fat; n−3, omega-3. Results are representative of 2 independent experiments. Significance determined by t test (phyla) or ANOVA with Bonferroni’s correction (genera): * = p value <0.05, ** = <0.01, *** = <0.001, – = not significant. The results from the Low Fat group shown in this table are a subset of what was previously reported in reference 13. The remaining data is novel.</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 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 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 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