Characterization and complete genome of the virulent Myoviridae phage JD007 active against a variety of Staphylococcus aureus isolates from different hospitals in Shanghai, China

A total of 175 isolates of S. aureus were obtained from Ruijin Hospital, the Sixth People’s Hospital of Shanghai, the Armed Police General Hospital, and the Centre Hospital of Changning District in Shanghai, China. The strains isolated from Ruijin Hospital were kindly provided by Qingtian Li, Those from the Sixth Hospital by FeifeiGu and Lizhong Han, those from the Armed Police General Hospital by Yunheng Zhou, and those from the centre hospital of Changning District in Shanghai, China were by Ren Wang. The use of these isolates in this paper was approved by these individuals, the ownership of the strains individually belonged to them, and we were authorized to use these isolates in this paper. The isolates were grown in liquid LB (Luria-Bertani) medium at 37 °C, on solid LB medium (1.5% agar), or in LB soft agar overlays (0.7% agar). Phage JD007 was isolated from chicken faeces collected from the chicken slaughter facility in the Madang food market located on No.349-2# Madang Road in the Huangpu District of Shanghai, China [17]. S. aureus strain Sa60 isolated from Ruijin Hospital was used for phage JD007 amplification and the following experiments. Forty-one strains of the 175 total strains represent different types of S. aureus identified and characterized using MLS and Spa typing methods, as described previously [22]. Briefly, the strains were spa-typed via the online database (http://​www.​spaserver.​ridom.​de/​). The sequence type (ST) was characterized by multi-locus sequence typing (MLST), and the products of seven house-keeping gene fragments were sequenced (Sangon Biotech, Shanghai) and compared to allele profiles from the database of S. aureus (http://​saureus.​mlst.​net/​). All of the bacteria were identified using the VITEK2 compact system, and antimicrobial susceptibility testing was performed by the disk diffusion method according to Clinical and Laboratory Standards Institute guidelines (M100S, 26^thedition) for penicillin (P) (10 units), cefoxitin (30 μg), gentamicin (CN) (10 μg), erythromycin (E) (15 μg), teicoplanin (30 μg), clindamycin (CC) (2 μg), rifampicin (5 μg), and linezolid (30 μg). The minimum inhibitory concentration (MIC) of vancomycin was determined using an E-test. S. aureus ATCC25923 and ATCC29213 were used as quality controls for the disk diffusion test and MIC detection, respectively.

High-titre phage stocks were obtained through amplification in liquid LB medium containing 10 μM MgCl₂ and 5 μM CaCl₂. First, phage JD007 that infected S. aureus Sa60 cells at an MOI of 0.1 was incubated at 37 °C overnight. Visible lysis of the liquid culture was obtained, and the lysate was then incubated with chloroform (final concentration of 2%) for 30 min with gentle shaking to kill the remaining bacteria. Bacteria debris was removed by centrifugation at 6,500 rpm (Beckman, JA18.0, USA) for 15 min. The phages in the supernatant were enriched at 4 °C overnight using polyethylene glycol (PEG) 8000 and precipitated (final concentration 10%w/v) at 8,500 rpm for 20 min (Beckman, JA18.0). The pellet was dissolved in TM buffer and vortexed. PEG8000 was removed after adding the same volume of chloroform after vortexing, and the solution was centrifuged at 4,000 × g for 10 min, the supernatant contained a high concentration of phage. CsCl was added at a concentration of 0.5 g per 1 mL, and the phages were purified by discontinuous centrifugation through a CsCl gradient (1.33, 1.45, 1.50, and 1.70 g/cm³) in TM buffer in Ultra-Clear tubes (Beckman Coulter, Inc., Fullerton, CA) at 120,000 × g for 4 h. The band of enriched phages was removed using a syringe, and the same was dialyzed against TM buffer and stored at 4 °C.

Using the purified phage obtained above. Then, phage particles were collected by centrifugation at 33,000 × g for 1 h and washed twice in 0.1 × PBS (pH7.4) using a Beckman high-speed centrifuge and a JA-18.1 fixed-angle rotor. Following deposition onto a carbon-coated copper grid and staining with 2% (wt/vol) potassium phosphotungstate (pH 7.0), the grids were observed using a Hitachi H7500 transmission electron microscope (TEM) operating at 80 kV.

The host range was analysed by spotting serial dilutions of phage JD007 on a double-layer soft agar lawn of different S. aureus isolates obtained from different hospitals. Two microlitres of concentrated phage lysate (≈10⁸ pfu/mL) and serial dilutions were plated onto LB medium plates overlaid with S. aureus (OD_600nm ≈ 0.4) mixed with 0.7% top agar (cultured for 30 min before adding), followed by overnight incubation. The strains with the same infectivity as the control strain (S. aureus Sa60) have an efficiency of plating of 1 (probably the equivalent of your “++++”). The inhibition zone of the spots formed by spot testing were seen as a common system for assessing the success of infection by the phage: ++++ complete clearing; +++ clearing throughout but with faintly hazy background; ++ substantial turbidity throughout the cleared zone; + a few individual plaques; 0 no clearing, − but you may see a spot where the pipette tip touched the agar [23]. In total, 175 isolates of S. aureus were used to identify the host range of phage JD007.

The complete genome sequence of JD007 has been reported, GenBank accession number is JX878671 [17]. It was sequenced using Roche 454 Sequencing Technology and assembled using the method of Newbler Metrics Results Software Release v. 2.7 the platform provided. All of the annotated genes were compared to an antibiotic resistance gene database (ARDB, http://​ardb.​cbcb.​umd.​edu/​) and a virulence factor database (http://​www.​mgc.​ac.​cn/​VFs/​main.​htm). Genes with more than 70% coverage and 30% identity were retained. The comparison of complete genome sequences of phage JD007 with phage K [24], Twort [4] and GH15 [25] using Mauve20150226 [26]. The maximum likelihood trees were constructed using MEGA5 [27].