Molecular Characterization and Genomic Analysis of Novel Phage vB_ ShiP-A7 Infecting Multidrug-Resistant Shiglla Fexneri and Escherichia Coli


 BackgroundPhage therapy has regained more attention due to the rise of multidrug-resistant (MDR) bacteria. Several case reports demonstrated clinical application of phage in resolving infections caused by MDR bacteria in recent years. ResultsWe isolated a new phage, vB_ShiP-A7, and then investigated its characteristics. Phage vB_ShiP-A7 is a member of Podoviridae that has an icosahedral spherical head and a short tail. vB_ShiP-A7 has large burst size and short replication time. vB_ShiP-A7’s genome is linear double stranded DNA composed of 40058 bp, encoding forty-three putative open reading frames. Comparative genome analysis demonstrated vB_ShiP-A7’s genome sequence is closely related to fifteen different phages (coverage 74-88%, identity 86-93%). Mass Spectrometry analysis revealed that twelve known proteins and six hypothetical proteins exist in particles of vB_ShiP-A7. Genome and proteome analyses confirmed the absence of lysogen-related proteins and toxic proteins in this phage. In addition, phage vB_ShiP-A7 can significantly reduce the growth of clinical MDR stains of Shigella ﬂexneri and Escherichia coli in liquid culture. Furthermore, vB_ShiP-A7 can disrupt biofilms formed by Shigella ﬂexneri or Escherichia coli in vitro. ConclusionPhage vB_ShiP-A7 is a stable novel phage, which has a strong application potential to inhibit MDR stains of Shigella ﬂexneri and Escherichia coli. Comparing the genomes between vB_ShiP-A7 and other closely-related phages will help us better understand the evolutionary mechanism of phages.


Introduction
Growing level of multidrug resistance (MDR) bacteria have been reported, and the emergence of these MDR bacteria leads to serious systemic and bio lm-associated infections [1,2,3]. For example, infection caused by MDR Enterobacteriaceae, especially the β-lactam resistance Enterobacteriaceae, is too hard to be treated [4]. Shigella species and Escherichia coli (E. coli) are major members of Enterobacteriaceae, which are important enteric pathogens [5,6]. In the era of the antibiotic crisis, bacteriophages have been studied as alternatives biocontrol agents for these members of Enterobacteriaceae [7].
Phage was rst used to treat dysentery caused by Shigella in 1920s [8]. After that, phages have long been used to treat dysentery in Eastern Europe [9]. In the 21st century, phages against MDR of Shigella have been investigated widely [10,11]. Shigella phage was successful applied to food safety [12]. Soffer et al. characterized ve lytic bacteriophages and combined them as a cocktail ShigaShield™, which could inhibit the growth of Shigellasonnei in food, in the process of FDA and USDA assessment for the GRAS status (GRN672) [13]. In mouse model, Mai et al. prepared a new reagent including phage cocktail and an antibiotic (ampicillin) named ShigActive™, which can inhibit Shigella effectively [14]. Bernasconi et al. proved three commercially available bacteriophage cocktails could suppress Shigella infections in human intestinal [15]. A cocktail including six lytic phages can inhibit Escherichia coli growth successfully [16]. A variety of mixed reagents including phages have been widely studied in the prevention and treatment of infections caused by Shigella species and E. coli in humans, suggesting that phages are promising for the treatment of infections caused by these members of Enterobacteriaceae.
Phage therapy needs appropriate lytic phages for different MDR bacteria. It has been shown that phage cocktail therapy can overcome the limitation of narrow host range of phage and phage resistance to bacteria [7,17]. Cocktails of well-known lytic phages might open new perspectives for successful controling MDR bacteria. A comprehensive study of each phage is also required to avoid phage-encoded toxic proteins or lysogen-related proteins. Therefore, isolating and well-characterizing more phages will allow us to get enough stock phages for selecting against different MDR clinical bacterial strains.
Phages have been isolated from different environmental sources and fecal samples of humans and other animals [18,19,20,21,22,23,24]. In this study, we isolated a lytic phage named vB_ShiP-A7 using MDR Shigella exneri as host from waste water in Nanjing, China. In addition, phage vB_ShiP-A7 can infect several clinically isolated MDR E. coli stains. Thus, this phage may be used to monitor, diagnose, and control infection caused by Shigella exneri and E. coli.

Bacterial strains
All the bacterial strains used in this study were all grown in Luria-Bertani (LB) medium at 37°C (Table 1). E. coli wild-type strain MG1655 was a stock of our lab. Shigella exneri A7, Shigella sonnei A5 and twenty-nine clinical stains of E. coli were isolated and cultured from different specimens of patients of the First A liated Hospital of Nanjing Medical University, Nanjing, China. Shigella exneri A7 was deposited in China Center for Type Culture Collection (CCTCC Number is PB 2020012) in Wuhan, China.
Isolation and propagation of bacteriophages vB_ShiP-A7 was screened from waste water in Nanjing (China) using multi-drug resistant Shigella exneri A7 (S. exneri A7) as host. Waste water samples were ltered through lters (0.45 μm Millipore, USA) rst. Then the ltered liquid was added to early-log-phase culture of S. exneri A7 and at cultured at 37°C for 4 hours to enrich phages. The cultures were spun down to removed bacterial cells. 10 μl supernatant, 100 μl S. exneri A7 and 3ml melted top agar mixed up well, then the mixture was poured on the surface of LB plate. After cultured at 37°C about 12 h, plaques formed on the plates. Single clear plaque was selected to start new round of screening. After several round screening, plaques were homogeneous on double layer agar plate. The preliminary puri ed phage from single plaque was got and kept at 4°C.

Puri cation of Bacteriophage vB_ShiP-A7
Bacteriophage vB_ShiP-A7 was puri ed following the protocol of Yu's [25]. Brie y, phage vB_ShiP-A7 were added into the early-log-phase liquid culture of S. exneri A7. Incubated at 37°C for another 2 h, the culture medium was spun down. The supernatant was collected and passed through lters (0.45-μm). The ltrate was concentrated by ultrahigh speed centrifugation. The supernatant was removed and the pellet was resuspended in SM buffer (10mM Tris-HCl, pH 7.5;100mM NaCl; 10mM MgSO 4 ). Further separation of the suspension by cesium chloride gradient ultrahigh speed centrifugation. We collected phage zone about 1 ml and diluted 10 times in SM buffer. Then the sample was participated at 200,000g for 3 hours to remove CsCl. The pellet was resolved in SM buffer, which is the puri ed phage particles of vB_ShiP-A7.
Electron microscopy A drop of ultracentrifuge purified Phage vB_ShiP-A7 particles was dripped onto a copper grid. The phages on the copper grid were negatively stained using 2% (w/v) phosphotungstic acid. The morphology of phage vB_ShiP-A7 were observed using FEI Tecnai G2 Spirit Bio TWIN transmission electron microscope at 80 kV.

Analysis of the phage host range
Infection ability of phage vB_ShiP-A7 on different strains were using the standard spot tests [26]. 100 μl of log-phage bacterial culture of each strain was mixed up with 3 ml of melted soft agar (0.6% agar), which was poured on top of LB plate. After we prepared different concentration of vB_ShiP-A7 phage suspensions (10 10 -10 2 pfu/ml), 5 μl of each concentration of the phage suspension were dropped onto the surface of the solidi ed plates containing different tested strains. After overnight cultured at 37 °C, the inhibition of bacterial growth by different concentration of vB_ShiP-A7 on each plate re ected the strain's sensitivity to vB_ShiP-A7. All experiments were conducted in accordance with the ethical rules of Nanjing Medical University (Nanjing, China) and the First A liated Hospital of Nanjing Medical University, and informed consents were obtained from all the patients.

Temperature Stability
The thermal stability of phage vB_ShiP-A7 was determined under different temperature. Five test tubes containing 10 9 phages were immersed in different temperature water bath about 1 hour (4°C, 25°C ,37°C, 45°C, 50°C). Then the phage titers of all the samples were determined by the double-layer method. Three independent repeated experiments were carried out. The average value was used to generate the gure and the standard deviation was marked.
One-step growth curve of phage vB_ShiP-A7 One-step growth curve of phage vB_ShiP-A7 was drew following the protocol of Yang's with minor modi cation [27]. Phage vB_ShiP-A7 was added to the early-log-phase of S. exneri A7 culture (1×10 8 CFU/ml) at a multiplicity of infection (MOI) of 10 and let them incubate with host strain for 10 min. After that, the phages were removed by centrifugation. The pellet was washed twice using fresh LB medium. Then, the precipitate was put into 50 ml LB medium and continue culture at 37°C. We got 1ml cell cultures at different time point and centrifuge at 14000 rpm for 1 minute remove the host bacteria. Free bacteriophage counts in these supernatants were counted using double-layer agar plate method. Three independent experiments were done to get the one-step growth curve of vB_ShiP-A7, in which the latency period, burst period and burst size of vB_ShiP-A7 were determined.

Bacterial challenge assay
Overnight culture of S. exneri A7 was inoculated into LB medium at a ratio of 1:100, and continue cultured for 2.5 hours to logarithmic phase. Phage vB_ShiP-A7 were added (MOI=10,1,0.1) to log-phage cultures of S. exneri A7. Bacterial culture was added with equal volume of SM buffer as negative control sample. Bacterial concentration was measured every 15 minutes for a total of 300 minutes.
Phage vB_ShiP-A7's genome isolation and sequencing The ultra-puri ed ShiP-A7's particles were digested by DNase I (New England Biolabs) and RNase A (Tiangen Biotech) at 37°C for 2 h to remove the residual genome DNA and RNA of host bacteria. Then, the sample treated by proteinase K (Tiangen Biotech) at 55°C for 15 min. Then this sample was further puri ed using TIANamp Bacteria DNA Kit (Tiangen Biotech). The puri ed Phage DNA concentration was measured by a spectrophotometer (Nanodrop Technologies, USA). The whole genome was sequenced on Illumina platform (Illumina HiSeq 2500 sequencer). SOAPdenovov2.04 software and GapCloserv1.12 was used to analyze high throughput sequencing results and assemble reads into a whole genome.

Annotation and comparison
Artemis software (http://www.sanger.ac.uk/science/tools/artemis) and Glimmer 3 [28] were used to nd putative open reading frames (ORFs) in vB_ShiP-A7's genome (The length of protein should not be less than 30 amino acids). Function annotation of vB_ShiP-A7's genome was conducted using the BLAST tools at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against the non-redundant protein sequences database. tRNAscan-SE was used to nd transfer RNAs (tRNAs) in vB_ShiP-A7's genome (v1.23, http://lowelab.ucsc.edu/tRNAscan-SE). RNAmmer was used to nd ribosome RNAs (rRNAs) in vB_ShiP-A7's genome (v1.2, http://www.cbs.dtu.dk/services/RNAmmer/). Molecular masses and isoelectric points of all the predicted phage proteins was calculated using DNAman. NCBI Megablast analysis was used to compare the whole genome sequence similarities of vB_ShiP-A7 with all the other bacteriophages. EMBOSS Needle tool was used to compare the similarity of protein amino acid sequences (European Molecular Biology Laboratory-European Bioinformatics Institute). EasyFig was used to compare annotated proteins of vB_ShiP-A7 with those of relative phages (http://mjsull.github.io/Easy g/ les.html) [29]. Neighbor-Joining algorithm in MEGA was used to analysis phylogenetic relationships among phages.
Analysis particles proteins of phage vB_ShiP-A7 vB_ShiP-A7's particles were mixed up with loading dye and boiled at 100 °C water bath for 5 min. The boiled sample was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel was stained with silver according to the protocol of Shevchenko's [30]. Liquid chromatography electrospray ionization with tandem mass spectrometry (LC-ESI MS/MS) was used to analysis proteins in vB_ShiP-A7's particles. vB_ShiP-A7 virions were digested with trypsin rst, then the tryptic peptides were analyzed by Q Exactive mass spectrometer (Thermo Scienti c, USA). MASCOT engine was used to nd the corresponding peptides (Matrix Science, London, UK; version 2.2) against all putative ORFs predicted in vB_ShiP-A7's genome.
Bio lm biomass quanti cation using crystal violet staining Overnight bacterial culture of S. exneri A7 was subcultured into LB medium until early mid-logarithmic phase (colony-forming units about 10 8 /ml). The bacterial culture was diluted to 10 7 /ml using LB medium. 200 µL/well of diluted bacterial cells (10 7 /ml) were added into 96-well plate (Corning Corp., United States). After incubated at 37 °C for 24 h, remove 100 µL of culture medium were removed from each well. At the same time, 100 µL fresh LB medium with phage vB_ShiP-A7 (10 9 /ml) were added into sample wells, and 100 µL LB were added in control wells. Continue to culture for another 24 h, culture mediums of each well were gently removed. Then washed the wells twice using 1×PBS. Bio lm attached to the wells was stained by 0.5% (w/v) crystal violet (200 µL/well) for 20 min at 37 °C. In order to remove the extra crystal violet stain, the wells were washed three times with PBS. The pictures of different wells were taken underneath Microscope.

Nucleotide sequence accession number
The whole assembled genome sequence of vB_ShiP-A7 is deposited at the GenBank database under accession number MK685668.

Morphology of Phage vB_ShiP-A7
Using S. exneri A7 E. coli as host strain, a novel phage vB_ShiP-A7 was isolated from waste water in Nanjing, China. After overnight incubation with S. exneri A7 at 37°C, phage vB_ShiP-A7 can form big clear round plague (diameter about 13 mm) on double-layer agar plate (Fig. 1A). We observed the morphology of this phage under Electron Microscope. vB_ShiP-A7 has isometric head with a mean diameter about 61.42+2.96 nm and noncontractile short tail about 13 nm in length (Fig. 1B). vB_ShiP-A7 is a member of viral family of podoviridae. This phage was named vB_ShiP-A7 followed the phage nomenclature de ned by Kropinski et al [31].
Thermal Stability and population dynamics of phage vB_ShiP-A7 The thermal stability of vB_ShiP-A7 was measured at different temperatures (4°C, 25°C, 37°C, 45°C, 50°C) ( Fig. 2A). The activity of phage vB_ShiP-A7 did not change much from 4°C to 37°C. When the temperature went up to 45°C, the phage started to lose their activity rapidly ( Fig. 2A). These data suggested that vB_ShiP-A7 is stable over a relatively wide temperature range from 4°C to 37 °C, and therefore, it can be preserved well at 4°C in the laboratory and play the role in human body at 37 °C.
One-step growth experiment was conducted to assess the population kinetics of vB_ShiP-A7 using strain S. exneri A7 as a host (Fig. 2B). vB_ShiP-A7 had been released 35 min after infection, with latent period about 30 min, and a burst size about 100 phage particles/cell (Fig. 2B).

Bacteriophage vB_ShiP-A7 inhibits planktonic bacterial growth
The e cacy of phage vB_ShiP-A7 on planktonic bacterial growth was assessed by inoculating a bacterial broth culture of S. exneri A7 with different multiplicity of infectivity (MOI, 0.1, 1 and 10) of phage vB_ShiP-A7. Our results showed that growth of host strain S. exneri A7 was completely inhibited within 90 min by phage vB_ShiP-A7 at different MOI, and this growth inhibition lasted until 300 min after infection (Fig. 2C). Compared to lower MOI (0.1, 1), higher MOI (10) of the phage can kill the host strain even faster (Fig. 2C).
Host range of phage vB_ShiP-A7 The ability of newly isolated phage vB_ShiP-A7 to infect different bacterial strains was estimated by the standard spot tests [26]. Phage vB_ShiP-A7 can infect MDR strain of S. exneri A7, but not MDR strain of S. sonnei A5 (Table 1). vB_ShiP-A7 can also infect three MDR E. coli stains isolated clinically, which can form clear plaque on two of the MDR E. coli strains and turbid plaque on another MDR E. coli strain at low concentration of phage (Table 1). In addition, vB_ShiP-A7 can't infect wild-type E. coli stain MG1655 (Table 1), which means it may not affect the normal ora of human. Thus, vB_ShiP-A7 may be used as biocontrol agents to prevent or treat infection caused by MDR S. exneri or E. coli.

Basic characteristics of vB_ShiP-A7 genome
To exclude the possibility that vB_ShiP-A7 contains any virulent proteins, it is necessary to understand complete genome sequence of vB_ShiP-A7. Next generation sequencing results suggested that the complete genome of phage vB_ShiP-A7 is a linear double-stranded DNA about 39881 bp. There is a single cut site of PstI near one termini of phage vB_ShiP-A7's genome and a single cut site of EcoRI near the other termini of phage vB_ShiP-A7's genome. After digesting the phage genome with PstI or EcoRI and re-sequencing the small fragments of these two enzymes' digestion products, we proved that the genome DNA of vB_ShiP-A7 contains 177 bp terminal repeats locating at the genome from nucleotides 1 to 177 and 39882 to 40058, respectively (Fig. 3). Therefore, the nal genome length of phage vB_ShiP-A7 is 40058 bp with 49.4 % GC content (Table 2, Fig. 3). The general organization of vB_ShiP-A7's genome follows that of T7-like phages, in which forty-three putative open reading frames (ORFs) were predicted in complementary strand (Table 2). We did not nd tRNA genes and rRNA genes in vB_ShiP-A7's genome.
The annotated 43 ORFs were summarized in Table 2. Twelve hypothetical proteins are predicted in vB_ShiP-A7's genome. Functions of these hypothetical proteins in vB_ShiP-A7 life cycle need to be determined in the future studies. Thirty-one ORFs (72.1%) were highly homologous to known functional genes, which were predicted to have similar functions with related genes (Table 2), and labeled in different colors in Fig. 3. The predicted functional proteins encoded by vB_ShiP-A7 can be divided into ve categories: DNA/RNA replication/modi cation (DNA polymerase, DNA primase/helicase, ssDNAbinding protein, DNA ligase, RNA polymerase, bacterial RNA polymerase inhibitor, nucleotide kinase, exonuclease, endonuclease), host lysis (lysin protein, endopeptidase Rz), packaging (DNA packaging protein, DNA packaging protein A), structural proteins (tail ber protein, internal virion protein D, internal core protein, DNA injection channel protein A , internal virion protein A, tail tubular protein B , tail tubular protein A, major capsid protein, capsid and scaffold protein, head-to-tail joining protein, tail assembly protein, host range protein), and additional functions (carbohydrate ABC transporter permease, Nacetylmuramoyl-L-alanine amidase, dGTP triphosphohydrolase inhibitor, putative protein kinase, putative S-adenosyl-L-methionine hydrolase, predicted antirestriction protein ) (Table 2, Fig. 3). In addition, lysogen related proteins, such as integrase, recombinase, repressor and excisionase, were not presented in vB_ShiP-A7's genome. We believe that phage vB_ShiP-A7 is a lytic bacteriophage. We did not observe any poison proteins encoded by vB_ShiP-A7's genome. The characteristics of this lytic phage without harmful factors encoded make it an ideal antibacterial agent.
We compared vB_ShiP-A7 with its 15 related phages using EasyFig. Most of the proteins encoded by vB_ShiP-A7 and its 15 related phages are highly similar (Fig. 5). Several dissimilarity proteins among these phages are showed in blank or light color in Fig. 5. DNA ligase encoded by ORF36 shows a divergence among these relative phages (100% coverage, 65-74% identity). Tail assembly protein encoded by ORF15 is different from the homologous proteins of other related phages (45-96% coverage and 57-89% identity). Tail ber protein encoded by ORF5 of vB_ShiP-A7 has relatively lower homology with its related phages (37-46% coverage and 54-60% identity) (Fig. 5). The similarity of vB_ShiP-A7's tail ber protein with other homologs is only found at the N-terminus, which is associated with the tail structure [35]. The C-terminus of this tail ber protein, involved in ligand interactions, exhibits relatively large variability with tail ber proteins of related phages (Fig. 5).

Structural Proteins of vB_ShiP-A7
Puri ed phage vB_ShiP-A7's particles were denatured and separated by SDS-PAGE. At least eight distinct protein bands were shown in the silver-stained SDS-PAGE gel, with molecular weights ranging from 17 to 180 kDa, (Fig. 6). Seven bands were speculated as structural proteins of vB_ShiP-A7 by estimated molecular weights (internal virion protein D, tail tubular protein B, tail ber protein, head-to-tail joining protein, major capsid protein, capsid and scaffold protein, tail tubular protein A) (Fig. 6). To further con rm these structural proteins, phage vB_ShiP-A7' particle proteins were determined by mass spectrometry (Table 3). Total eighteen proteins were identi ed, including all the proteins showed on SDS-PAGE gel (Table 3, Fig. 6). Nine of them are known structural proteins. DNA primase/helicase was also determined in the phage particles. In addition, some hypothetical proteins (ORF17, ORF18, A7_225, A7_120, A7_146, A7_426, A7_68, A7_88) were also detected by Mass spectrometry, and their functions need to be determined further. Hypothetical proteins encoded by A7_225, A7_120, A7_146, A7_426, A7_68, A7_88 were only predicted when we used all the possible ORFs by artemis in vB_ShiP-A7' genome as reference (>30 amino acids). But they were omitted from annotation le of vB_ShiP-A7 (uploaded to NCBI under assigned number MK685668), since they don't have similar sequences with any predicted proteins at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) or their genes may exist in the interior of known genes. Interestingly, A7_225, A7_120, A7_146, A7_68 and A7_88 were encoded by antisense RNAs on known genes of late operon of vB_ShiP-A7 (Table 3). A7_225 was translated on the opposite direction of ORF30 (endonuclease). A7_120 was existed on the opposite direction of ORF11 (tail tubular protein A). A7_146 was existed on the opposite direction of ORF14 (head-to-tail joining protein). A7_68 was existed on the opposite direction of ORF6 (internal virion protein D). A7_88 was translated on the opposite direction of ORF7 (internal core protein). In addition, no toxic protein was identi ed by Mass spectrometry in vB_ShiP-A7's particles.

Ability of vB_ShiP-A7 to destroy bacterial bio lms
Removal of bio lm is the key to treat chronic infectious diseases. We tested the effect of phage vB_ShiP-A7 on bio lm formed by S. flexneri A7 and E. coli 395 B5, respectively. About 24 h post phage vB_ShiP-A7 addition, bio lm biomass of strain S. flexneri A7 (Fig. 7C) and E. coli 395 B5 (Fig. 7D) were signi cantly lower to that of the untreated controls (Fig. 7A, B). This analysis suggested that vB_ShiP-A7 can reduce the bio lm formation on clinic strains of Shiglla flexneri A7 and E. coli 395 B5, raising a possibility of using phage vB_ShiP-A7 as a bio lm disruption agent.

Discussion
Shigella species and E. coli belong to Enterobacteriaceae, which can cause intestinal infection [6]. Of note, previous studies have demonstrated that bacteriophages can be used to treat this infection [15,16]. In this study, a new lytic phage vB_ShiP-A7 was isolated and characterized, with lytic activity against both MDR S. exneri clinical strain and several MDR E. coli clinical strains (Table 1). Several phages were reported to have the ability to infect both E. coli and Shigella [40,41,42]. Since E. coli and Shigella are genetically similar, it is reasonable to expect some similar phages would target both strains. In addition, lytic phages are relative specific, and usually infect a subgroup of strains within one bacterial species or across closely-related species, which causes less disruption to gut ora than antibiotic treatment [14]. In addition, Phage vB_ShiP-A7 does not infect wild-type E. coli strain MG1655, suggesting that it may not affect the normal ora.
Phage vB_ShiP-A7 belongs to family Podoviridae according to its morphology under the Electron Microscope (Fig. 1). vB_ShiP-A7 has short latent time and a large burst size, suggesting that vB_ShiP-A7 can quickly increase the phage concentration [43]. Next-generation sequencing demonstrated that the genome of phage vB_ShiP-A7 does not encode integrases, recombinases, or harmful gene products (  Fig. 3). In addition, phage vB_ShiP-A7 has shown promising effects against bacterial growth in liquid and bio lm (Fig. 2, Fig. 6), suggesting that it may be used as an ant-infective agent.
Comparative genome analysis demonstrated that phage vB_ShiP-A7 is related to unclassi ed T7-like phages (Fig. 4, Fig. 5). Therefore, this phage can be assigned into virulent phage of T7-like family. Only several genes of vB_ShiP-A7 genome are dissimilar with their relative phages (Fig. 5), in which the tail ber protein of vB_ShiP-A7 is obviously different with that of other phages. The tail protein of phage is the key protein to recognize host bacteria. Different tail proteins of phages can cause phages to infect different host bacteria [44]. Yosef et al reported that a small change in the tail protein sequence of a phage can lead to changes in host range [45]. The different tail ber protein of vB_ShiP-A7 may allow this phage to infect some speci c hosts which could be used as a component of phage cocktail. Most of the genes of vB_ShiP-A7 and 15 related phages are highly homologous. High homology of same functional phage genes was found in different phage species, illustrating that horizontal gene transfer between phages is a component of evolution [32,45]. Gene arrangement of phage vB_ShiP-A7 is different with some of related phages (Fig. 5). Gene rearrangement was also observed in other Escherichia phages [47]. vB_ShiP-A7 and its relatives may be evolved through horizontal exchange and rearrangement of their genes, which is a common phenomenon in the evolution of tailed phages [32,35,38].
Eighteen proteins were identi ed in vB_ShiP-A7 phage particles using mass spectrometry, including known structural proteins and hypothetical proteins ( Table 3, Fig. 6). Interestingly, some of these hypothetical proteins were encoded by antisense RNA on late operon encoded structure proteins (A7_225, A7_120, A7_146, A7_68, A7_88) ( Table 3). Anne et al found PAK_P3 expressed antisense RNA elements targeting its structural region during the early stage of infection [48], which might be used to shutdown expression of late structural genes during the early stage of infection. An antisense RNA was also found in lambda phage genome, which was transcripted from paQ promoter and didn't encode protein or peptides [49]. We found several small peptides/proteins encoded by antisense RNAs in E. coli phage vB_EcoP-EG1 [50]. These small peptides/proteins encoded by antisense RNAs in late operon may exit in different phages and involve in the phage infection processes, however the detailed mechanisms remain to be determined.
The phage genome lays a foundation for studying the interaction between phage and its host. Comparative genome analysis of this phage with related phages shed light on the mechanisms of evolutionary changes of these T7-like family phage genomes. Fully characterized phage could be used as alternative treatment for the increasing number of MDR Shigella and Escherichia, and hence reducing the pressure to nd new antibiotics.