Lysozyme

Lysozyme was purchased from Sigma-Aldrich (dialyzed and lyophilized 629710). Initial protein solutions were prepared by solving lysozyme in sodium acetate buffer (50 mM sodium acetate, pH 4.4) prepared with Milli-Q water (Millipore, Billerica, MO, USA). Prior to use, the lysozyme solutions were prepared using lysozyme, crystallized and dialyzed four times, either through a preparative column or filtered through Amicon ultra filters, in order to remove any unwanted aggregates present in most of the commercially available lysozyme preparations [33 (link)]. Water-in-oil droplets of protein solution at different concentrations (by on-chip dilution of a mother solution using acetate buffer) were generated and transported by an immiscible fluorous oil (Krytox GPL100, DuPont, Wilmington, DE, USA) containing 2% w/w of fluorinated surfactant (tri-block copolymer, PFPE-PEG-PFPE, purchased from RAN Biotechnologies, Beverly, MD, USA) in order to stabilize the droplets interface. The selection of both oil and surfactant was made considering a compromise between good resistance to X-ray radiation damage, an optimal viscosity, and immiscibility with the aqueous phase, as previously reported [11 (link)].

Pelleted bacterial cells were resuspended in 200 μl of lysozyme/mutanolysin (LM) solution (1xTE buffer (pH 8.0) containing 15 mg/ml lysozyme (Sigma, Germany) and 500 U/ml mutanolysin (Sigma, Germany)) ([30 (link)]). The cell suspension was vigorously shaken for 45 min at 13.000 rpm using an Eppendorf shaker. Afterwards the suspension was transferred to a 15 ml falcon tube containing 50 mg of 0.1 mm zirconia beads (Roth, Germany) and 700 μl RLT lysis buffer (Qiagen, Germany). After vigorous vortexing for 3 min, the beads were removed by centrifugation (11.000 g, 2 min, RT). 470 μl of 100 % ethanol (Roth, Germany) were added and thoroughly mixed with the supernatant which was then applied in two steps to the Qiagen Spin Column. The subsequent RNA extraction procedure was performed according to the manufacturer’s instructions, including the on-column DNase I digestion with the Qiagen DNAse I kit.

At 17 weeks of age, the kittens were housed individually for 24 h and a fresh faecal sample was collected from each animal within 15 min of excretion, snap-frozen in liquid nitrogen and stored at −85 °C6 (link). High molecular weight metagenomic DNA was extracted from faecal samples using a non-mechanical lysis method as follows. One hundred mg of faecal material was incubated in 300 μl of a lysozyme and RNAse solution (40 mg lysozyme [Sigma L6876; Sigma-Aldrich, St. Louis, MO, United States] + 1 ml 10 mM Tris HCl/1 mM EDTA + 15 μl RNAse cocktail [Ambion AM2286; Thermo Fisher Scientific, Waltham, MA 02451, USA]) for 30 min at room temperature. Afterwards, 60 μl of Proteinase K solution (Ambion AM2546) was added to each tube and incubated for a further 30 min at room temperature. Extraction of DNA was completed using Nucleospin Soil kits (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions.

To assess the effects of reduced osmolarity in 0.1BHI on viability of bacteria, aliquots of the preculture used for biofilm assays were diluted 1:100 in either 0.1BHI or demineralized H₂O (dH₂O) and viable cell counts were determined as colony‐forming units per ml (CFU/ml) by spot‐plating. For this purpose, 10 µl aliquots of 10‐fold serial dilutions were plated in triplicate onto BHI agar and the colonies of an appropriate dilution were counted to calculate CFU/ml. The effect of lysozyme treatment was analyzed in a similar assay except that bacteria were inoculated from a preculture into 0.1BHI, grown at 25°C to exponential growth phase (OD_600nm = 0.15–0.2), harvested by centrifugation and resuspended in 0.1BHI containing 5 µg/ml lysozyme (40,000 Units/mg, Sigma‐Aldrich). Bacteria were incubated in the presence of lysozyme at 25°C for the indicated time and log‐reduction was calculated relative to CFU/ml at t = 0 min of an untreated control, that is, an aliquot resuspended in 0.1BHI without lysozyme.

C. perfringens strain SM101, grown under identical conditions in BHI alone or BHI containing nisin and bile acids, was used to isolate RNA. The RNA was extracted from the cells according to a method previously described [25 (link)]. Cells harvested at various intervals after incubation were used for RNA extraction. The cells were centrifuged (15,000 ×g, 10 min, 4°C), washed with 10 mM Tris and 1 mM EDTA (pH 8.0), and suspended in lysozyme buffer [25 (link)] containing 10 mg/ml of lysozyme (Sigma). Following incubation for 10 min at room temperature, the cells were centrifuged (15,000 ×g, 10 min, 4°C). The cell pellets were suspended in 50 μl of TE (10 mM Tris, 1 mM EDTA) and mixed with 350 μl of RNAWIZ from Ambion (Grand Island, NY). 75 μl of chloroform was added to the samples, which were incubated for 30 min on ice. The samples were then centrifuged (15,000 ×g, 30 min, 4°C) and the clear phases containing RNA were removed to different tubes. The RNA samples were applied to an RNeasy Mini Kit from Qiagen, Inc. (Valencia, CA), to further purify RNA. Contaminating DNA was removed using a Turbo DNA-free kit (Ambion). A Nanodrop ND-1000 spectrophotometer (NanoDrop Technology, Wilmington, DE) was used to determine the quantity and quality of total RNA. To avoid RNA degradation, the RNA was stored at −80°C and used for qRT-PCR within a week of extraction.

To extract DNA from each culture sample, 5 μl of the collected aliquot was mixed with 1 μl lysozyme solution (50 mg/ml lysozyme [Sigma], 20 mM Tris–HCl [pH 8.0], 2 mM EDTA), and the mixed solution was incubated at 37°C for 2 h. After adding Proteinase K solution (1/30 [v/v] Proteinase K [Takara], 20 mM Tris–HCl [pH 8.0], 2 mM ethylenediaminetetraacetic acid (EDTA)), the aliquot was incubated at 55°C for 3 h and 95°C for 10 min. The solution was then vortexed for 10 min to increase DNA yield.
We also extracted DNA from the inoculum aliquot to reveal community structure of the source microbiome. Because the source inoculum was expected to include high concentrations of soil-derived compounds, which can inhibit polymerase chain reactions (PCRs), a commercial DNA extraction kit optimized for soil samples was used. Specifically, after 50 μl of the inoculum sample was incubated with 350 μl SDS buffer with Proteinase K (1:30 [v/v] Proteinase K [Takara], 0.5% sodium dodecyl sulfate (SDS), 2 mM Tris–HCl [pH 8.0], 2 mM EDTA) based on the temperature profile of 55°C for 180 min and 95°C for 10 min. The aliquot (400 μl) was then subjected to DNA extraction with DNeasy PowerSoil Kit (Qiagen).