1, CU459141.1, and
CP001182.1). Random amplification of polymorphic DNA (RAPD) analysis was subsequently used to discriminate the A. baumannii strains. Primers Wil2 (Williams et al., 1993) and 1247 (Akopyanz et al., 1992) previously used for typing other bacteria were applied. Some other representatives of the genus of Acinetobacter such as A. lwoffii (six strains), A. anitratus (4), and A. calcoaceticus (3) and several other gram-negative microorganisms such as P. aeruginosa, Escherichia coli, Yersinia pseudotuberculosis, Yersinia enterocolitica, Klebsiella pneumoniae, Ruxolitinib purchase Klebsiella oxytoca, Enterobacter cloacae, Pasteurella multocida, and Salmonella Enteritidis (three strains of each species) were used in the research. All bacteria were grown in Luria–Bertani (LB) broth or nutrient agar (Himedia Laboratories Pvt. Limited, India) at 37 °C. Clinical materials and in-hospital environmental samples were used for phage isolation. Nonliquid samples were kept in 0.1 M Tris–HCl buffer, pH 7.0. The samples were cleared by low-speed centrifugation (7000 g for 30 min.) followed by filtration of the supernatants through 1.20- and 0.45-μm-pore-size membrane filters (Millipore) to remove bacterial debris. The purified filtrates were concentrated by ultracentrifugation at 85 000 g at 4 °C for 2 h (Beckman SW28 rotor). The spot test method as well as the plaque assay (Adams, 1959) was used to screen for the presence of lytic
phage activity RGFP966 cell line in the resultant concentrates using clinical A. baumannii strains of different RAPD groups. The plates were incubated overnight at 37 °C and examined for zones of lysis or plaques formation. Single plaque isolation was used to obtain pure phage stock. For that a single plaque formed on the A. baumannii lawn was picked
up in SM buffer (10 mM Tris–HCl, pH 7.5, 10 mM MgSO4 × 7 H2O, and 100 mM NaCl) and replated three times. Phage AP22 was propagated using liquid culture of identified A. baumannii clinical strain 1053 (OD600 nm of 0.3) at multiplicity of infection (MOI) of 0.1. The incubation was performed at 37 °C until complete lysis, Methisazone and then chloroform was added. Bacterial debris was pelleted by centrifugation at 7000 g for 30 min. The phage lysate was concentrated by ultracentrifugation at 85 000 g at 4 °C for 2 h (Beckman SW28 rotor). The resultant pellet was carefully mixed with SM buffer and centrifuged at 13 000 g. Supernatant was treated with DNase (1 μg mL−1) and RNase (1 μg mL−1) at 37 °C. The nucleases were removed with chloroform. The phage preparation with the titer of 1012–1013 PFU mL−1 was purified by cesium chloride equilibrium gradient centrifugation at 100 000 g (Beckman SW41 rotor) for 24 h (Sambrook et al., 1989). Host specificity of the phage was determined by double-layer method. Onto the surface of M9 medium (Sambrook et al., 1989) plates, 0.3 mL of liquid bacterial culture (108–109 PFU mL−1) and 4 mL of soft agar (LB broth supplemented with 0.