0.22 m pore size filter

Roswell Park Memorial Institute 1640 (RPMI) medium (with L-glutamine, and pH indicator, but without bicarbonate) (Merck, Darmstadt, Germany) supplemented with dextrose to a final concentration of 2%, buffered with MOPS (Merck) at a final concentration of 0.165 mol/L, and adjusted to pH 7.0 with 1 M sodium hydroxide was used as a test medium. The medium was prepared at double strength to allow a two-fold dilution. After preparation, the medium was sterilized by vacuum filtration through a 0.22 µm pore size filter (Merck).

Virus-encoded BiKEs were purified from supernatants of infected Vero cells (vBiKEs). 6 × 10⁶ cells in 15 cm culture dishes were inoculated with respective MV-BiKEs at MOI 0.03 in OptiPRO SFM serum-free medium (Thermo Fisher Scientific), cultured at 37 °C, 5% CO₂ and monitored for syncytia formation. Supernatants were collected, clarified (2500 × g, 5 min, 4 °C), and passed through a 0.22 µm pore size filter (Merck) before vBiKEs were purified by affinity exchange chromatography via the His₆ tag. Ni-NTA Spin Columns or Superflow Columns (Qiagen) were used for small and large scale purification, respectively, following the manufacturer’s instructions. In brief, cell-free supernatants were applied to equilibrated columns allowing BiKE binding to the Ni-NTA resin. Columns were rinsed with wash buffers containing 10 mM and 20 mM imidazole. Immobilized BiKE proteins were recovered with elution buffer containing 500 mM imidazole. Eluates were washed with DPBS, concentrated in Amicon Ultra-15 Centrifugal Filter Units with Ultracel-10 membranes (Merck), and stored at −80 °C. Protein concentration in purified vBiKE aliquots was quantified by BCA assay using the Novagen BCA Protein Assay Kit (Merck) according to the manufacturer’s instructions.

Vesicles were isolated according to published protocol [33 ]. Briefly, E. coli O83 was grown in BHI in an overnight culture at 37 °C and 180 rpm. The main culture was inoculated 1:100 from the overnight culture and grown for 8 h at the same conditions. The culture was centrifuged for 15 min at 4 °C and 10 000 × g. The supernatant was filtered through a 0.22 µm-pore-size filter (Merck Millipore, Burlington, MA). OMVs were isolated by ultracentrifugation (4 h at 4 °C and 150 000 × g; Beckman Coulter 45Ti rotor). The pellet was dissolved in 500 µl of sterile 0.9% NaCl (B. Braun, Maria Enzersdorf, Austria). OMVs were stored at 4 °C for immediate use or at -20 °C for long-term storage.

The antagonistic activities were measured by the Oxford cup method as described previously [12 (link)]. Pathogenic bacteria were used as follows: Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 29213, Salmonella typhimurium (S. typhii) ATCC 14028, and Streptococcus agalactiae (S. agalactis) ATCC 13523. Moreover, Lactobacillus rhamnosus (L. rhamnosus) ATCC 7469 was used as a contrast. Briefly, the Lactobacillus strains were cultured overnight in MRS broth at 37 °C in a shaker. Pathogenic bacteria were cultured in LB (Luria Bertani) broth at 37 °C in a shaker for 24 h. The LAB strains were centrifuged at 6000× g for 10 min and the supernatant was collected and filtered using a 0.22 µm-pore-size filter (Millipore). Approximately 10⁷ CFU/mL of each pathogen strain was spread onto the surface of LB agar plates. Wells of diameter 5 mm were punctured into the inoculated pates, and 100 µL of supernatant were added into each well and cultured at 37 °C for 24 h. Then, the inhibition zone diameters were measured using a vernier caliper. The experiments were performed in triplicate. The strains with antibacterial activity were used for the subsequent assay.

Cell-free culture supernatant was prepared as previously described [115 (link)], with modifications. Briefly, LF3872 was grown overnight in MRS broth under anaerobic conditions at 37 °C. The overnight culture was diluted to the concentration of 1 × 10⁸ CFU/mL in MRS broth and further grown anaerobically for 48 h. Cell-free culture supernatant was collected by centrifugation at 5000× g for 20 min at 4 °C; filter-sterilized using a 0.22 µm pore size filter (Millipore, Billerica, MA, USA); and concentrated by speed-vacuum drying (Rotational Vacuum Concentrator RVC2-18, Martin Christ, Osterode, Germany). The lyophilized sediment of CSLF3872 alone or together with the prebiotic was used to study the effectiveness of inhibiting the adhesion of proteobacteria pathogens (E. coli, Salmonella, and Campylobacter).

Native cell-free supernatant (CFS) was prepared from cultures of the LS7247 strain as previously described in [113 (link)] with modifications. Briefly, the LS7247 strain was grown for 18 h in MRS broth under anaerobic conditions at 37 °C. The culture was diluted to a concentration of 1 × 10⁸ CFU/mL in MRS broth and further grown anaerobically for 48 h. CFS was collected by centrifugation at 6000× g for 25 min at 4 °C, filter-sterilized using a 0.22 µm pore size filter (Millipore, Bedford, MA, USA), and concentrated by speed-vacuum drying (Rotational Vacuum Concentrator RVC2-18, Martin Christ, Osterode am Harz, Germany). To obtain the ∆CFS, proteinase K (1.0 mg/mL) was added to native CFS from LS7247 and incubated at 37 °C for 2 h.
Native CFS and ∆CFS from the LS7247 strain alone or together with the Actigen prebiotic (Alltech Inc., Nicholasville, KY, USA) were used to study the effectiveness of inhibiting the adhesion of SE and ST pathogens to human and animal enterocytes.

Virus lipid labeling using 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl)oxacarbocyanine (SP-DiOC18; Molecular Probes catalog no. D7778) and octadecyl rhodamine b chloride (R18; Molecular Probes catalog no. O246) was described previously (25 (link), 26 (link)) and was adapted to the single use of SP-DiOC18 or 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate (DiD; Molecular Probes catalog no. D7757). In brief, IAV (PR8M) was labeled with SP-DiOC18 and R18 at final concentrations of 0.2 µM and 0.4 µM, respectively, or with SP-DiOC18 or DiD alone at 0.2 µM for 1 h at room temperature and was subsequently filtered through a 0.22-µm-pore-size filter (Millipore). Cells were infected with the labeled virus for 30 min on ice, washed twice with PBS, and subsequently incubated for the indicated periods of time at 37°C. Cells were harvested by Accutase (Millipore catalog no. SCR005) treatment and were fixed with 2% formaldehyde for 20 min. Lipid mixing was quantified by FACS analysis, with 10,000 cells analyzed per sample. Linear regression analysis (GraphPad Prism) was used to determine the best-fit linear regression and to calculate the slopes.