Lysozyme

Cells were harvested and DNA was extracted as previously described by Siebert et al. [24 (link)]. In brief, 30 mL bovine raw milk were treated with 1.8 mL 0.3 M EDTA. After the cell harvesting by centrifugation (20 min at 4 °C) and the removal of the milk fat and skimmed milk in the supernatant, the selective lysis of the somatic DNA was performed using proteinase K (20 mg/mL, AppliChem GmbH, Darmstadt, Germany) and DNase I (Thermo Fisher Scientific, Waltham, MA, USA). The DNA extraction was performed with the PowerFood Microbial DNA isolation kit (Qiagen, Hilden, Germany), modified by an additional enzymatic lysis step. Towards this end, lysozyme (25 µg/mL, Carl Roth) and mutanolysin (100 U, Sigma-Aldrich, St. Louis, MO, USA) were added to the cell suspensions together with the MBL solution of the DNA isolation kit, followed by an incubation at 37 °C and 350 rpm for 30 min. After an additional treatment with proteinase K (12.5 mg/mL, AppliChem), the remaining bacterial cells were disrupted in tubes with silica beads using a FastPrep-24TM instrument (MP Biomedicals, LLC, Irvine, CA, USA). The subsequent DNA isolation followed the manufacturer’s protocol, i.e., that of the PowerFood Microbial DNA isolation kit. The DNA was finally eluted in 2 × 24 µL of PCR-grade water (preheated to 55 °C).

Bacterial cultures were prepared as described for FISH. After drying on a glass slide, the bacteria were permeabilized with lysozyme (Carl Roth, 10 mg/ml) for 7 min and afterwards rinsed shortly with water. Samples were then blocked with blocking buffer [2 % of bovine serum albumin in PBS (Sigma-Aldrich, USA)] for 1 h at room temperature. Subsequently, the primary antibody [anti-(TEM) β-lactamase ab12251 (mouse); abcam, United Kingdom], diluted 1:200 in blocking buffer, was added and incubated either overnight at 6 °C (for sequential stainings) or 1 h at room temperature. Slides were rinsed shortly with water and washed three times with PBS and, finally, with blocking buffer for 3 min each. Slides were then incubated with the secondary antibody (goat anti-mouse IgG-Alexa Fluor® 488 ab150117, abcam), diluted 1:300 in blocking buffer, for 1 h at room temperature. Slides were again rinsed shortly with water and washed three times with PBS for 3 min each, once more rinsed with water, air-dried and embedded in Roti®-Mount FluorCare DAPI. Each bacterial strain was stained in three independent immunofluorescence assays.

Biopsies from the HSCT CD cohort were resuspended in 180 μl TET (TrisHCl 0.02M, EDTA 0.002M, Triton 1X) buffer and 20mg/ml lysozyme (Carl Roth, Quimivita, S.A.). Samples were incubated for 1h at 37°C and vortexed with 25 μl Proteinase K before incubating at 56°C for 3h. Buffer B3 (NucleoSpin Tissue Kit–Macherey-Nagel) was added followed by a heat treatment for 10 min at 70°C. After adding 100% ethanol, samples were centrifuged at 11000 x g for 1 min. Two washing steps were performed before eluting DNA. Concentrations and purity were checked using NanoDrop One (Thermo Fisher Scientific). Samples were immediately used or placed at -20°C for long-term storage.