Bacterial strains, plasmids, and growth conditions
E. 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 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 bacteriophages
Bacteriophage P954 is a temperate phage that was isolated from 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). The first fragment (bp 1-376) was amplified with forward primer 5'-CGGAATTCcatatgAAAACATACAGTGAAGCAAGAGCA-3', containing an NdeI restriction site, and reverse primer 5'-CCGCCGCTgaattcTAATAAAGTGAGTACAGCC-3', containing an EcoRI site. The fragment was cloned into a pET21a vector at the NdeI/EcoRI sites.
The second fragment (bp 377-753) was amplified with forward primer 5'-CCGCCGGgaattcAGTATAAAAGTGAGGGCTTA-3', containing an EcoRI site, and reverse primer 5'-CCaagcttTTAAAACACTTCTTTCACAATCAATCTCTC-3', containing a HindIII site. The second fragment was cloned in tandem with the first fragment, thus generating the full-length phage P954 lysin gene with an internal EcoRI site. The cat gene was isolated along with its constitutive promoter from the S. aureus - E. coli shuttle plasmid pSK236 by ClaI digestion. Cohesive ends were filled with the Klenow fragment of DNA polymerase I and ligated into the blunted EcoRI site of the full-length phage P954 endolysin gene, thereby disrupting it. The S. aureus-specific temperature-sensitive origin of replication from the shuttle vector pCL52.2 was introduced at the XhoI restriction site of this construct to generate pGMB390.
Mitomycin C induction of phage P954 lysogens
The S. aureus RN4220 lysogen of phage P954 was inoculated in LB medium and incubated at 37°C with shaking at 200 rpm for 16 hr. The cells were then subcultured in LB medium at 2% inoculum and incubated at 37°C with shaking at 200 rpm until the culture attained an absorbance of 1.0 at 600 nm. Mitomycin C was then added to a final concentration of 1 μg/ml, and the culture was incubated at 37°C with shaking at 200 rpm for 4 hr for prophage induction.
Recombination and screening for recombinants
S. aureus RN4220 cells were transformed with pGMB390 by electroporation according to the protocol described by Schenk and Laddaga  with a BioRad Gene Pulser, plated on LB agar containing chloramphenicol (10 μg/ml), and incubated at 37°C for 16 hr. Chloramphenicol-resistant colonies were selected and grown in LB at 37°C until the cultures reached an absorbance of 1.0 at 600 nm. Recombination was then initiated by infecting these cells with phage P954 (MOI = 3) for 30 min. Progeny phage were harvested from the lysate as described previously, lysogenized in S. aureus RN4220, and plated on LB agar containing chloramphenicol (10 μg/ml) (round I). Ninety-six chloramphenicol-resistant colonies were picked up, grown, and induced with Mitomycin C. Cultures that did not lyse after the 16-hr Mitomycin C induction were treated with 1% chloroform and lysed with glass beads; the released phages were again lysogenized in S. aureus RN4220 (round II). Chloramphenicol-resistant colonies of round II lysogens were similarly grown and subjected to Mitomycin C induction. The chloramphenicol-resistant lysogens that did not release phages upon Mitomycin C induction were selected for PCR analysis. Genomic DNA of the selected lysogens was purified, and PCR was performed with different sets of primers to confirm disruption of the phage P954 endolysin gene.
Endolysin complementation for phage enrichment and enumeration
The endolysin gene from a Podoviridae phage in our collection, P926, was cloned under the constitutive B. 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 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 . 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, 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 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 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 7.5 (negative control); groups 2 and 5 were treated with two doses of endolysin-deficient phage P954 prepared in 25 mM Tris-HCl, pH 7.5 at 200 MOI equivalent (MOI relative to CFU at LD80); and groups 3 and 6 were treated with two doses of chloramphenicol (50 mg/kg). The first treatment dose was administered immediately after challenge; the second dose was administered 2 hr later. Mice were observed over 10 days for occurrence of mortality. Survival analysis is plotted as Kaplan-Meier survival curves using MedCalc statistical software version 184.108.40.206 (Mariakerke, Belgium).