subtilis vegII promoter in an E. coli – S. aureus shuttle vector constructed in our laboratory. This construct, designated pGMB540, was used for trans-complementation of the selleck screening library nonfunctional endolysin for propagation of the recombinant phage in lytic mode and for their enumeration. Plasmid pGMB540 was introduced into S. aureus strain RN4220 by electroporation according to the protocol described by Schenk and Laddaga [30]. Transformants

were selected on LB medium containing tetracycline (5 μg/ml) and used as bacterial hosts for phage enrichment. Early log phase cells of S. aureus RN4220/pGMB540 grown at 37°C were infected with the recombinant endolysin-deficient phage P954 (MOI = 0.1) and incubated for an additional 3 to 4 hr until the culture lysed. The phage-containing lysate was passed through a 0.2-μm filter, Combretastatin A4 order and the phages were enumerated on a lawn of S. aureus RN4220/pGMB540 cells. The endolysin-deficient phage P954 was also enriched by induction. Briefly, the lysogen was grown at 37°C until absorbance at 600 nm reached 1.0 and then induced with 1 μg/ml Mitomycin C at 37°C for 4 hr. The cells were pelleted and lysed by vortexing with glass beads. Cell selleck chemical debris was removed by centrifugation at 5000 × g for 10 min, and the phage-containing supernatant was passed through a 0.2-μm filter. Comparison of in vitro bactericidal activity of parent and lysis-deficient phage P954 The parent and

recombinant phages were compared for host range and bactericidal activity. Ten MOI equivalent of phage was added to 2 × 108 colony-forming units per ml (CFU/ml) and incubated at 37°C for 90 min. Serial 10-fold dilutions of the mixture were plated on LB agar, and residual viable cells (CFUs) were enumerated. In vivo efficacy of endolysin-deficient

phage P954 in neutropenic mice Animal experiments were performed at St. John’s Medical College and Hospital, Bangalore, India. The experiments were approved by the Institutional Animal Ethics Committee and the Committee for the Purpose of Control and Supervision of Experiments on Animals (registration No. 90/1999/CPCSEA dated 28/4/1999). Healthy male Swiss albino mice (6-8 weeks old, neutropenic) were used to evaluate in vivo efficacy. Neutropenia was induced by intraperitoneal (IP) administration of cyclophosphamide (100 mg/kg). In Sclareol a preliminary study, the lethality of a clinical MRSA isolate (B911) was determined in the mice (1 × 107 -1 × 108 CFU). We found that 5 × 107 CFU resulted in 80% mortality (LD80), and it was therefore chosen as the challenge dose to evaluate phage efficacy (data not shown). In the efficacy experiment, mice were assigned to six treatment groups (n = 8, each group). Four days after cyclophosphamide treatment, the mice in groups 1-3 were challenged with B911 (200 μl, 5 × 107 CFU). Groups 1 and 4 were then treated with 25 mM Tris-HCl, pH 7.