0.1 mm glass beads

Once obtained during endoscopy, tissue biopsies were placed into sterile Powerbead tubes pre-loaded with 0.1 mm glass beads (Qiagen, Germantown, MD) plus external lysis buffer in vitro diagnostic (200 µL, Roche Applied Science, Indianapolis, IN). Tissues were homogenized at 30 Hz for 5 min using a Tissuelyser II homogenizer (Qiagen). Swabs from the uvula were placed into sterile PBS, vortexed and then were aliquoted 1:1 into the external lysis buffer (100 µL). Sample lysates were deposited into individual wells of 96 deep-well processing plates. DNA was subsequently extracted in high-throughput fashion using a MagNA Pure 96 instrument running a DNA and viral small volume-in vitro diagnostic extraction kit according to the manufacturer’s protocol (Roche). After extraction, a portion of the DNA was evaluated by Ion Torrent Next Generation Sequencing or using the EMB. The remaining material was archived at −20 °C.

For Lactobacillus whole cell assays, cells were centrifuged and washed twice with sterile PBS, pH 7.4, then resuspended in fresh media. T-β-MCA (Steraloids) was added to a concentration 500 nM, and cultures were incubated at 37°C for 90 mins. This concentration was chosen as it provided visible peaks by GCMS analysis and also allowed us to visualize concentration changes over time (see UPLC-ESI-QTOFMS analysis below). Cells were precipitated by centrifugation and 100 μL of supernatant was added directly to 100 μL of ice cold methanol and stored at -80°C for analysis by UPLC-ESI-QTOFMS. Cell lysate assays were used to assess BSH substrate specificity in transgenic E. coli C600 strains and were performed by washing overnight cultures twice in sterile PBS, pH 7.4, and resuspending the cells in 3 mM sodium acetate buffer, pH 5.2. Cells were lysed by vortexing with 0.1 mm glass beads (Mo Bio Laboratories, Inc.) according to the manufacturer’s instructions. Total protein was measured by Bradford assay and normalized to 0.100 mg/mL in 100 μL of buffer before adding a concentrated master solution of T-β-MCA, TCA, TDCA, TCDCA, GCA, and GCDCA (Sigma) to achieve a final concentration of 500 nM. Cell lysates were incubated at 37°C for 90 mins, quenched with 100 μL of ice cold methanol and stored at -80°C for analysis by UPLC-ESI-QTOFMS.

A 350 μL aliquot of the homogenized sample was mixed with 0.9 volume of MagNA Pure Bacterial Lysis Buffer (Roche Applied Science, Indianapolis, IN), lysozyme (final concentration, 2.9 mg/mL; Sigma-Aldrich Corp., St. Louis, MO), and lysostaphin (final concentration, 0.14 mg/mL; Sigma-Aldrich), then incubated for 30 min at 37°C[23 (link)]. Samples were transferred to a tube containing 0.1 mm glass beads (MoBio Laboratories, Carlsbad, CA) and agitated in a Mini-Beadbeater-9 (Biospec Products Inc., Bartlesville, OK) for 1 min at the maximum setting. Samples were digested with Proteinase K (final concentration, 1 mg/mL) and incubated for 10 min at 65°C, agitated for an additional 1 min in the Mini-Beadbeater-9, and then incubated for an additional 10 min at 95°C. DNA purification was performed using an automated nucleic acid purification platform (MagNa Pure Compact System, Roche) using the manufacturer’s DNA Bacteria v3.1 protocol.