Fastprep 24 5g

Fecal samples were homogenized using the FastPrep-24 5G (4m/s, Adapter: QuickPrep, Time: 5 sec, Cycle:1, Lysing Matrix: D) (MP Biomedicals) in PBS containing 0.1% Tween 20 (10mg/ml) and stored at −20°C until analysis. Once thawed, samples were centrifuged at 20,000 rpm for 5 minutes to clarify and then supernatants were serially diluted (1:2, 1:20, 1:200) for analysis using the Mouse Lipocalin-2/NGAL DuoSet ELISA kit (R&D SYSTEMs) according to manufacturer’s instructions. A standard curve was generated and concentrations of Lcn-2 per gram of feces was calculated by linear regression analysis (GraphPad Software).

Wild type and mutant S. aureus strains were grown overnight at 37°C with shaking in 15 mL conical tubes containing 5 mL of RPMI + BCFA medium. 50 μL of these overnight cultures was inoculated into 15 mL conical tubes containing 5 mL of RPMI + BCFA; RPMI + BCFA + 5 μM α-lipoic acid, or RPMI + BCFA + 150 μM octanoic acid. Samples were incubated with shaking at 200 rpm for 9 hours and bacterial growth was determined by measuring OD600 using a Genesys 10S UV-Vis spectrophotometer (Thermo Scientific). The remaining culture volume was centrifuged at 3,000 x g for 15 minutes, the supernatant was discarded, and the bacterial pellets were suspended in 250 μL of PBS and transferred to screw cap microcentrifuge lysing tubes (Fisher Scientific) containing 250 μL of 0.1 mm glass cell disruption beads (Scientific Industries, Inc.). Cells were lysed using a Fast Prep-24 5G (MP Biomedicals) bead disruption system in two sequential steps, at 5.0 speed for 20 seconds and at 4.5 speed for 20 seconds, each separated by a 5 minute incubation period on ice. After cell disruption, samples were centrifuged at 19,000 x g for 15 minutes. 45 μL of the supernatant were collected in microcentrifuge tubes containing 15 μL of 6X SDS sample buffer and subsequently boiled for 10 minutes prior to storage at −20°C.

Genomic DNA was extracted using the PowerLyzer Power-Soil DNA Isolation Kit (MO BIO Laboratories, Inc.; Carlsbad, CA, USA) and following the manufacturer’s instructions. For air/dust samples, 125–250 µl of particulate matter were loaded into a bead tube. For soil samples, ~0.75 g of soil were loaded into a bead tube. The FastPrep-24 5G (MP Biomedicals, LLC.; Santa Ana, CA, USA) high-speed benchtop homogenizer was used for optimal homogenization and cell lysis. Samples were homogenized for seven 1-min cycles at 6 m/s with 5-min rest breaks between each cycle. Upon addition of elution buffer to the spin filter, samples sat at RT for 5 min before centrifugation.