Tryptic soy agar

Yeast extract and curcumin were obtained from Acros Organics (Geel, Belgium). Tryptic soy agar and gold(III) chloride trihydrate were acquired from MP Biomedicals (Santa Ana, CA). Ampicillin, isopropyl β-D-1-thiogalactopyranoside (IPTG), imidazole, sodium monobasic phosphate, sodium dibasic phosphate, sodium dodecyl sulfate, sodium hydroxide, sodium chloride, sucrose, tris-hydrochloride, tryptone, PFU high fidelity, DpnI, ACS grade methanol and urea were obtained from Fisher Scientific (Pittsburgh, PA). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), magnesium sulfate, nickel chloride, sodium borohydride were purchased from Sigma Aldrich (St. Louis, MO). Tricine was purchased from Alfa Aesar (Ward Hill, MA). Glacial acetic acid and Factor Xa cleavage kit were purchased from EMD Millipore (Rockland, MA). Ethyl acetate was purchased from Pharmco-AAPER (Brookfield, CT). Ethylenediaminetetraacetic acid (EDTA) and hydrochloric acid were acquired from VWR (Radnor, PA). HPLC grade methanol was obtained from Ricca Chemical Company (Arlington, TX). Sephadex™ G-25 medium beads were purchased from Amersham Pharmacia Biotech AB (Piscataway, NJ). Columns were purchased from Bio-Rad (Hercules, CA).

Quantification of the total number of adherent and internalized bacteria was determined at 1, 4, 8, 12, 16, 20, and 24 h post-bacterial infection. Respiratory epithelial HPAEpiCs were harvested for bacterial enumeration by washing 2× with DPBS followed by gentle scraping. Cells were pelleted by centrifugation at 5000 rpm for 10 min, supernatant removed, and cells resuspended in 0.025% TritonX-100. Colony forming units (CFU) were quantified by standard bacterial plating on tryptic soy agar (MP Biomedicals, LLC, Solon, OH, USA). Four biological replicates were performed at each time point for enumeration. RT-qPCR was used to quantify viral replication by collecting supernatant samples for IAV–MRSA infected alveolar epithelial cells. Total RNA was extracted from the supernatant using the PureLink Viral RNA/DNA Mini Kit (LifeTechnologies, Burlington, ON, Canada) according to the manufacturer’s instructions. Reverse transcription of total RNA was performed using the Superscript IV first-strand cDNA synthesis kit (Life Technologies, Burlington, ON, Canada) using primers specific for the viral H1N1 HA sequence. Viral genome copy numbers were quantified by comparing RT-qPCR results to an established external viral genome copy number standard.