coli strain DH5α [Φ80dlacZΔM15 Δ (lacZYA-argF) recA1 endA1 hsdR17 supE44 thi-1 gyrA96relA1deoR] was used as host for plasmid constructions and plasmid propagation. A restriction-deficient prophage-free S. aureus strain RN4220  was used for recombination, lysogenization, and phage enrichment. Clinical isolates of S. aureus were used to test phage sensitivity. A MRSA clinical isolate (B911) was used in animal experiments to determine the in vivo efficacy of the endolysin-deficient phage P954. The plasmid pET21a (Novagen, USA) was used for cloning and construction RG7112 purchase of endolysin disruption
cassette. The plasmid pSK236, an E. coli – S. aureus shuttle vector containing pUC19 cloned into the HindIII site of S. aureus plasmid pC194 , was used as a source for the cat gene. A shuttle vector containing the temperature-sensitive replication origin of S. aureus, pCL52.2, was used as source for the replication origin . The constitutive
Bacillus subtilis vegII promoter was derived from pRB474 . All bacterial strains were cultured in liquid Luria Bertani (LB) medium at 37°C on a rotary shaker (200 rpm) unless otherwise stated. Ampicillin, chloramphenicol, and tetracycline were used as needed. All chemicals were obtained from Sigma-Aldrich, St. Louis, MO, USA unless otherwise mentioned. Propagation, concentration, and enumeration of SCH727965 bacteriophages Bacteriophage P954 is a Saracatinib cell line temperate phage that was isolated from
Venetoclax mouse the Ganges River (India) and amplified in S. aureus strain RN4220. Briefly, S. aureus RN4220 was grown at 37°C in LB medium to an absorbance of approximately 0.8 at 600 nm, infected with phage P954 at a multiplicity of infection (MOI) of 0.01, and cultured at 37°C until the culture lysed completely. After centrifugation at 4100 × g for 10 min to remove cell debris, the bacteriophages were concentrated by centrifugation at 27,760 × g for 90 min. The bacteriophage titer was determined by enumerating plaque-forming units (PFUs) in serial 10-fold dilutions in LB medium and confirmed by the agar overlay method [27, 28]. Preparation of phage P954 DNA and genome sequencing Phage P954 DNA was prepared from a stock solution (1 × 1012 PFU/ml). The concentrated phage preparation (1 ml) was incubated at 37°C for 1 hr with DNase I (1 μg/ml) and RNase A (100 μg/ml). The mixture was adjusted to contain 1% sodium dodecyl sulfate, 50 mM EDTA (pH 8.0), and 0.5 μg proteinase K and incubated at 65°C for 60 min. The mixture was then subjected to phenol-chloroform-isoamyl alcohol (25:24:1) extraction, and the DNA was precipitated . Purified phage DNA was used for genome sequencing [GenBank: GQ398772]. Construction of plasmids for phage P954 endolysin disruption The phage P954 endolysin gene (753 bp) was amplified as two separate fragments by polymerase chain reaction (PCR).