Lysostaphin

The stool samples were thawed and archaeal DNA was extracted from 150 mg of each patient’s stool sample. We used a universal method developed by us to extract the DNA of microorganisms (both bacteria and archaea). Isolation was performed, using the Genomic Mini AX Stool (A&A Biotechnology, Gdynia, Poland) along with pretreatment, including enzymatic lysis using lysozyme (A&A Biotechnology), lysostaphin (A&A Biotechnology) and mutanolysin (A&A Biotechnology), followed by mechanical lysis using a FastPrep homogenizer (MP Biomedicals). Briefly, 150 μl NaCl, 20 μl of lysozyme, 10 μl of lysostaphin and 5 μl of mutanolysin were added to the tubes containing feces and glass homogenizer beads. The samples were homogenized (30 s, speed 4.0 m/s) using FastPrep and incubated for 30 min at 37°C. The consecutive steps of DNA isolation were performed according to the Genomic Mini AX Stool protocol pursuant to the producer’s instructions. After isolation, DNA concentration and purity were verified spectrophotometrically using a NanoDrop instrument (Thermo Fisher Scientific, Boston, MA, USA).

A 2 mL sample of overnight bacterial culture in BHI broth (BTL, Warsaw, Poland) was centrifuged at 12,000× g for 5 min. The bacterial pellet was suspended in 150 μL of 0.1 M Tris-HCl buffer, pH 7.4, containing 2 U of lysostaphin (A&A Biotechnology, Gdańsk, Poland), and incubated for 30 min at 37 °C. Next, 15 μL of 10% SDS was added and incubated for 15 min at 37 °C, and then 200 μL of 5 M guanidine hydrochloride solution was added and left for 10 min at room temperature. The DNA was extracted by phenol and chloroform, precipitated by ethanol, dissolved in 50 μL of UltraPure™ Distilled Water (Thermo Fischer Scientific Inc., Waltham, MA, USA) and stored at −20 °C.

Two-millilitre aliquots of an overnight bacterial culture in BHI broth (BTL, Poland) were centrifuged at 12,000 × g for 5 min. The bacterial pellet was resuspended in 150 μl 0.1 M Tris–HCl buffer, pH 7.4, containing 2 units of lysostaphin (A&A Biotechnology, Poland), and incubated at 37 °C for 30 min. Then 15 µl of 10% SDS was added and incubated at 37 °C for 10 min, followed by the addition of 200 µl of 5 M guanidine hydrochloride solution and incubation for 10 min at room temperature. DNA was extracted by phenol and chloroform, precipitated by ethanol, dissolved in 50 μl of UltraPure™ Distilled Water (Thermo Fischer Scientific Inc., USA) and stored at − 20 °C.

Plasmid DNA was isolated from Listeria spp. and B. subtilis strains, as described previously [23 (link)]. In the case of S. aureus strains, lysostaphin (A&A Biotechnology, Gdańsk, Poland) was used instead of lysozyme (Sigma-Aldrich). Plasmid DNA for sequencing was isolated using large-scale preparation methods [80 (link)], and sequence assembly was verified by RFLP analysis. Purified plasmid DNA was digested with restriction enzymes EcoRI, BamHI or NcoI, according to the manufacturer’s protocol (Thermo Scientific, Waltham, MA, USA). The resulting DNA fragments were separated by electrophoresis in 0.8% agarose gels.

The bacterial cells were grown in 20 mL BHI broth to logarithmic stage (OD_600nm = 0.6), and harvested by centrifugation. The cell pellet was suspended in 20 mL of 4% SDS, and inactivated at 65°C for 2 h. The inactivated cell suspension was centrifuged, and the cells were washed with deionized water three times. The washed cell pellet was suspended with PBS to reach 0.4 OD_600nm, and then tested for susceptibility to commercially available lysostaphin (E. coli recombinant, Prospec, Ness-Ziona, Israel) or mutanolysin (recombinant, A&A Biotechnology, Gdynia, Poland) lytic enzyme. lysostaphin and mutanolysin were added to 1 mL of the tested cell suspension at a final concentration of 8 μg/mL and 0.2 U/mL, respectively, followed by incubation in a plastic cuvette at 30°C at 120 rpm rotation. The reduction in OD by cell lysis was measured using a DU730 spectrophotometer (Beckman Coulter, CA, United States).