5% SDS, 20 mM EDTA, and 50 μg mL−1 proteinase К at 56 °C for 3 h. The DNA was extracted with phenol/chloroform (1 : 1) and then precipitated with ethanol supplemented with sodium acetate. Restriction fragment length Bleomycin molecular weight polymorphism (RFLP) analysis of the phage DNA was performed using endonucleases AluI, ApaI, BamHI, BglII, CfrI, ClaI, DraI, DraII, Eco52I, EcoR91I, EcoRI, EcoRV, HindIII, HinfI, MspI, NcoI, NheI, NotI, PstI, PvuII, RsaI, SalI, SmaI, SmiI, SspI, TaqI, VspI, and XmiI (Fermentas, Lithuania).
The procedure was carried out according to instructions provided by the manufacturer. DNA fragments were separated by 0.8% and 1.5% agarose gel electrophoresis in TBE electrophoresis buffer. The approximate molecular sizes of separated DNA fragments were calculated using Quantity One software (Bio-Rad). One-kb DNA Ladder (Fermentas) and phage lambda DNA digested with HindIII were used as molecular markers.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed according to the Laemmli’s standard protocol (Laemmli, 1970) using the CsCl-purified phage preparation. The phage was examined by negative contrast electron microscopy (EM; Brenner & Horne, 1959). The purified and concentrated virus preparation was fixed with 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) and then placed on transmission Doramapimod chemical structure EM support grids followed by rinsing with distilled water several times. The phage samples were stained with 1% uranyl acetate aqua solution for further examination with a Hitachi H-300TM electron microscope (Japan). Electron microscope magnification was calibrated using T4 phage as size standard. At least 20 electronic phage images were used for phage morphology determination.
To obtain electron microphotographs of the phage–host cell interaction, the mixture of A. baumannii 1053 cells (107 CFU mL−1) with the phage AP22 (108 PFU mL−1) was incubated in 0.05 M phosphate buffer (pH 7.0) for 10 min at room temperature. The phage–cell mixture was fixed with 1% glutaraldehyde pentoxifylline in 0.1 M phosphate buffer (pH 7.0) and analyzed as described earlier. The problem of the search for potentially therapeutic A. baumannii phages and their characterization has recently attracted considerable attention because of the increasing interest of the microorganism itself as a threatening causative agent of nosocomial infections worldwide. Phage AP22 was isolated from clinical materials provided by the burn center of N.V. Sklifosovsky Scientific Research Institute of First Aid (Moscow). A phage was isolated from the burn center in a month and found to be identical in host range and RFLP assays with AP22. It can be assumed that the bacterial virus is stable in the hospital environments. The phage AP22 formed clear, round plaques of 2–3 mm in diameter with haloes (zone of clearance around each plaque) on A. baumannii-sensitive strains.