Biological characterization and genomic analysis of a novel methicillin-resistant Staphylococcus aureus phage, SauPS-28

ABSTRACT Staphylococcus aureus, a representative gram-positive bacterium, is a common infectious pathogen widely present in the natural environment. The increasing application of antibiotics is witnessing an increment in the number of clinically resistant strains (such as methicillin-resistant S. aureus [MRSA]), which has posed a great challenge to antimicrobial therapy. In this study, a novel MRSA phage, SauPS-28, was isolated from the lake water of the Guangxi Zhuang Autonomous Region. This phage has an incubation period of approximately 30 min, a lysis period of approximately 40 min, and a burst size of approximately 25 PFU/cell. The isolated phage exhibited good biological stability at a pH range of 6.0–9.0 and temperature range of 4°C–37°C. In addition, the identification of an elongated tail using transmission electron microscopy confirmed that SauPS-28 belongs to the long-tailed phage family. Whole-genome sequencing analysis revealed that SauPS-28 has a 43,286-bp-long genome with 31.03% G + C content. Moreover, SauPS-28 exhibited 95.69% sequence identity with ECel-2020k, while the query coverage was only 66%, which is a newly discovered phage. Whole-genome functional annotation results revealed that SauPS-28 had 68 open reading frames (ORFs). Of these, 30 ORFs are unknown proteins. The results suggest that SauPS-28 could be a lysogenic phage strain. This study thus provides preliminary data to conduct further in-depth analysis of the mechanism of phage–host interaction and provides a reference value for phage therapy. IMPORTANCE In recent years, drug-resistant bacterial infections have become increasingly serious. As a kind of virus with the ability to infect and lyse drug-resistant bacteria, phage is expected to be a new therapeutic method. In this study, we isolated and purified a new methicillin-resistant Staphylococcus aureus bacteriophage SauPS-28, studied a series of biological characteristics of the bacteriophage, analyzed the genome and structural proteome data of the bacteriophage, and provided reference data for further study of the interaction mechanism between bacteriophage and host bacteria and promoted new antibacterial strategies.

Phages are a class of viruses with prokaryotic bacterial hosts and are characterized by high diversity, abundance, and specificity (12)(13)(14).They are categorized as lysogenic and lytic.In 1921, phages were first discovered to possess the ability to lyse bacteria and were therefore used to treat bacterial infections (15).The discovery and extensive use of antibiotics thereafter contributed to the decline in phage therapy research.However, the recent increase in cases of antibiotic resistance in bacteria and the completely different bactericidal mechanisms from that of antibiotics have renewed interest in phage therapy (16)(17)(18).Lysogenic phages are diverse and widely distributed, with nearly half of the sequenced bacteria being lysogenic (19); however, their ability to kill bacteria is limited, and superinfectious immunity caused by gene integration also reduces the susceptibility of host bacteria to phages (20).Horizontal gene transfer (HGT) causes toxin genes, virulence island gene clusters, and antibiotic-resistant genes in bacteria, which make them more virulent and difficult to treat (21)(22)(23)(24)(25)(26)(27), so the first choice for phage therapeutic recommendations are strictly lytic phages that can kill the host quickly and effectively.However, this guideline was developed before efficient strategies of phage genome engineering.As high-throughput sequencing and synthetic biology advanced, the special advantages of lysogenic phages were gradually revealed.The rapid increase in the number of bacterial genome sequences in bioinformatics databases, the development of bioinformatics tools for genomic analysis and lysogenic phage marker gene (integrases, etc.) identification, and the abundance of lysogenic phages in bacterial genomes have made the detection and isolation of lysogenic phages easier, in particular for anaerobic and in vitro difficult-to-incubate pathogenic bacteria (28).Lytic mutant phage variants can be obtained by the mutagenesis of lysogenic phages and gene editing techniques, which could remove gene modules known to be involved in the establishment and maintenance of the lysogenic life cycle and genes for bacterial virulence in lysogenic phages, and then transform lysogenic phages into strictly lytic phages (29,30).Lysogenic phages can have their host range and bactericidal potential altered by genetic engineering and recombination procedures (29).The use of designed lysogenic phages to transport synthetic gene networks that interfere with crucial bacterial intracellular processes has been made possible by advances in synthetic biology (31).These synthetic gene networks can cause cell death of the bacteria or convert them to be antibiotic sensitive (32).The focus on lysogenic phage utilization will help contribute to the development of more comprehensive bacterial treatments.
Several successful cases of phage application in models or clinical treatments have been reported, which fully validate the feasibility of phage therapy (33)(34)(35)(36)(37).For instance, Doub et al. and Schoeffel et al. reported the effectiveness of S. aureus phages in the treatment of recalcitrant MRSA-induced arthritis (34,38).Teng et al. evaluated the effectiveness of phage treatment for mastitis in a mouse model of MRSA and demon strated that MRSA colonization in the mammary gland was significantly inhibited (39).Chung et al. reported that lysogenic phage D3112 significantly reduced mortality in fly models infected with Pseudomonas aeruginosa either orally or via injection (40).Breyne et al. adopted a phage cocktail method to treat bovine mastitis and found a significantly reduced infection rate of bovine mastitis in mice, with improved pathologic changes and reduced bacterial counts (41).Meanwhile, phage antibiotic combination (PAC) therapy has become a research hotspot (42).Rodriguez et al. successfully cured MRSA-induced refractory chronic sinusitis (43).Both phage therapy and PAC therapy have shown the potential value of phages against drug-resistant bacterial infections, such as MRSA infections.
In this study, a novel strain of MRSA phage was isolated and identified from Guangxi Zhuang Autonomous Region.Preliminary studies on the biological properties of this novel strain, such as lysis ability, growth curve, and stability, were conducted, and preliminary analyses of the whole genome and proteome were conducted.

Morphological observation of phage SauPS-28
A novel MRSA phage, SauPS-28, was isolated from the lake water of Guangxi Zhuang Autonomous Region, China, by using MRSA obtained from the Second Affiliated Hospital of Guilin Medical College as the host strain.Clear and round phage spots were observed on the double-agar plate (DAP), confirming the lytic ability of SauPS-28 (Fig. 1A).The morphological features of this phage were observed under a Hitachi TEM HT7700 at 80 kV.SauPS-28 was tadpole-shaped with a positive polyhedral head structure, approxi mately 40 nm in diameter.It had an approximately 190-nm-long slender tail and an overall length of approximately 230 nm, which was typical of T-series phages (Fig. 1B).According to the classification guidelines proposed by the International Committee on Taxonomy of Viruses, the morphology of phage SauPS-28 was consistent with the morphological characteristics of Siphoviridae: it is thus a new member of the Siphoviri dae family (44).

Host-range determination
SauPS-28, 22 MRSA strains, and the host range of SauPS-28 were detected using the DAP method (Table 1).Excluding the bacterial host, six MRSA strains were lysed by SauPS-28, and phage spots visible to the naked eye were formed on agar plates, which indicated that the phage had a wide host range.

Infection plural and growth curve
Most phage spots were observed on the DAP when SauPS-28 was mixed with the bacterial host at the optimal multiplicity of infection (MOI) ratio (Table 2).SauPS-28 produced the highest titer of daughter phages when it was mixed cultured with the host strain at the MOI ratio of 0.01.Three sets of parallel experiments were conducted for different MOI ratios, and the average of different results was used to plot the table.
SauPS-28 and the host strain were mixed at the MOI ratio of 0.01 and incubated at 37°C and 180 rpm, and the growth curve of SauPS-28 was plotted every 10 min by using the DAP method to detect the phage titer (Fig. 2).SauPS-28 entered the lytic phase after a latent period of approximately 30 min.The phage titer increased rapidly within 30-70 min, followed by a plateau phase, which indicated a lytic phase of approximately  Negative results are indicated as "N" and positive results as "Y".b 40 min.Phage burst is the ratio of the total daughter phage titer released from infected cells at the beginning of the plateau phase to the initial number of the infected host during the latent phase, with a burst of approximately 25 PFU/cell.The experiments were repeated thrice, and the error lines were presented as the mean ± SD.

Stability study of phage SauPS-28
SauPS-28 was incubated in different buffer pairs (pH = 5, 6, 7, 8, 9, 10, and 11) at 37°C for 1 h.The DAP method was used to assay the titers of phage suspensions at different pH values (Fig. 3).The optimum pH of SauPS-28 was 8, and the phage activity was completely lost at a pH below 5 or above 10.The experiment was repeated thrice, and the error line was presented as the mean ± SD.SauPS-28 thermal stability experiments were performed at different temperatures (4°C, 25°C, 37°C, 50°C, 60°C, and 70°C) and continuous incubation for 24 h.The DAP was used to assay SauPS-28 at different temperatures and different time points (0 min, 15 min, 30 min, 1 h, 2 h, 6 h, and 24 h), respectively, of phage titers (Fig. 4).The results revealed that SauPS-28 activity was stable at 4°C and 24 h at 37°C.At 50°C for 15 min, the titer of SauPS-28 decreased continuously, and SauPS-28 was completely inactivated at 6 h.At 60°C or 70°C, SauPS-28 was completely inactive.The experiment was repeated thrice, and the error line was presented as the mean ± SD.

Restriction digest profile of phage SauPS-28
The total nucleic acid of SauPS-28 was extracted using the Phage Genomic DNA Extraction Kit.Enzymatic identification was performed with restriction endonuclease EcoR I (Fig. 5).Lane M represents the marker, Lane 1 represents SauPS-28 total nucleic

Phage SauPS-28 proteome analysis
SauPS-28 was concentrated with PEG8000 and then, according to the BCA standard protein profile, ultrasonically broken and denatured at 100°C.SDS-PAGE was performed using Coomassie Brilliant Blue R-250 staining.The results revealed at least six more obvious protein bands in the SDS-PAGE gel (Fig. 6), corresponding to the results of functional annotation of open reading frames (ORFs) (Table 3).The predicted functions of the proteins encoded using the ORFs in the order of molecular weights from the largest to the smallest were 75.4 kDa (ORF29, encoding phage tail tape measure protein), 55.1 kDa (ORF16, encoding terminase), 43.7 kDa (ORF20, encoding phage major capsid protein), 38.8 kDa (ORF18, encoding phage portal protein), 36.4 kDa (ORF31, encod ing hypothetical protein), and 11.4 kDa (ORF22, encoding phage head-tail connector protein).Among them, ORF29, ORF20, ORF18, and ORF22 encoded structural proteins of phage SauPS-28, and ORF16 is a terminal enzyme.ORF31 is a hypothetical protein, and its potential function needs to be determined.

Genome annotation and function of the phage SauPS-28
Phage SauPS-28 has a linear double-stranded DNA with a 43,286-bp-long genome and a GC content of 31.03% (GenBank accession number: BankIt2665638 Seq1 OQ588745).The whole genome sequence of SauPS-28 was analyzed using BLASTn from the NCBI nonredundant DNA database.The whole genome sequence identity of SauPS-28 and phage ECel-2020k (CP062426.1)was 95.69%, while the query coverage was only 66%, thus indicating that the genome sequence of SauPS-28 was relatively new.Functional annotation using RAST revealed that SauPS-28 contained 68 ORFs.Of them, 53 ORFs had ATG as the start codon (77.94%), 9 ORFs had GTG as the start codon (13.2%), 5 ORFs had TTG as the start codon (7.3%), and 1 ORF had GTT as the start codon.A total of 61 ORFs were forward transcribed and 7 ORFs were reverse transcribed.The functional proteins encoded using the ORFs were analyzed using BLASTp and divided into six modules: virion structure, regulation, packaging, replication, lysis, and others (Table 3, Fig. 7).Some ORFs encoded proteins as part of the virion structure, including the phage protein, phage portal protein, phage major capsid protein, phage head-tail connector protein, and head-tail adaptor protein.Some ORFs encoded proteins involved in regulation (i.e., transcriptional regulator, Ig-like domain-containing protein, DUF2951 domain-contain ing protein, repressor-like protein, and helix-turn-helix domain-containing protein).Some ORFs encoded proteins involved in the packaging (i.e., HNH endonuclease, phage terminase small subunit, phage terminase, restriction endonuclease, and host-nuclease inhibitor Gam family protein).Some ORFs encoded proteins involved in replication, including nucleoside triphosphate pyrophosphohydrolase protein, site-specific inte grase, single-stranded DNA-binding protein, and ATP-binding protein).Some ORFs encoded proteins involved in lysis (i.e., holin and N-acetylmuramoyl-l-alanine amidase), which are responsible for most of the lysis of bacterial strains.The module categorized as other included the hypothetical protein, DUF3113 family protein, DUF1514 family protein, DUF2482 family protein, DUF1108 family protein, ERF family protein, DUF3269 family protein, DUF3113 family protein, as well as the toxin-antitoxin system, toxin component, MazF family, and phage prohead protease.This indicates that SauPS-28 is a lysogenic phage.The potential functions of hypothetical proteins in SauPS-28 need to be further investigated.No tRNA has been found in the SauPS-28 genome.

Phylogenetic tree analysis
To further analyze the evolutionary origin of SauPS-28, the whole genome sequence was analyzed using MEGA-X software to construct a phylogenetic tree (Fig. 8).According to the results, SauPS-28 belonged to the same group as phage ECel-2020n, phage ECel-2020p, phage vB SauS 308, and phage ECel-2020k, but in different branches.In addition, SauPS-28 was distant from all other phages.The results indicated that SauPS-28 was a new phage strain, and bootstrap values of >70 indicated reliable results.

DISCUSSION
The emergence of large numbers of multidrug-resistant bacteria poses a huge challenge to the treatment of bacterial infections, and phage therapy can be applied as the lastresort treatment as a compassionate medication approach when antibiotic therapies have demonstrated limited efficacy and it is difficult to reverse the disease (45,46).Lytic phages have a diverse host range that are optimal for phage therapy.In this study, we obtained the long-tailed phage SauPS-28 by isolation of lytic phages, and the sequenc ing results revealed a genome of length 43 kb.Homology comparison identified a 95.69% sequence identity with the whole genome of phage ECel-2020k, with 66% query coverage, which implies that it was a new phage virulence strain.Moreover, the gene annotation results indicated that it encoded at least 68 ORFs.The discovery of the gene encoding site-specific integrase suggests that it may belong to a lysogenic phage.Lysogenic phages can act as HGT vectors, and they act as a source of deleterious genes (e.g., virulence and antibiotic resistance) in the host bacterium through lysogeny, unlike lytic phages (21,(47)(48)(49).Therefore, performing the necessary genetic modifications and removing the corresponding deleterious genes is a prerequisite for lysogenic phage to use in phage therapy (48,50).Genetically modified lysogenic phages have been applied in the treatment of infections caused by Salmonella (51) and Mycobacte rium (52).In this study, experiments conducted on phage isolation, purification, multiplication, growth curves, and thermal and acid-base stability relied on plaque-forming units (PFU) counts (which are the typical characteristics of lysogenic phages) and revealed no obvious anomalies, possibly caused by the lysogenic phenomena.This observation is contrary to the results implied by the presence of site-specific integrases in the genome, with the following possibilities: (i) at the molecular level, the presence of a geneencoding integrase is not a sufficient condition for a phage to be identified as a lysogenic phage, and the sequencing results can only identify the presence of an integrase-coding sequence, which neither reflects the actual activity of integrase nor predicts the inevita ble occurrence of lysogeny; (ii) host bacteria and bacteriophages may lack all genetic elements or protein factors necessary for integration due to genetic mutations.Based on these factors, the categorization of phages as either lysogenic or lytic may be both crude and debatable, considering the changes in integrase activity potentially brought about through genetic mutations.Lysogeny or lysis (a phenomenon or state of affairs) is more likely to be host-dependent because of the coupling of a specific bacterium and a specific phage; however, the manifestation of host dependence may not be appropriate as a basis for phage categorization.Therefore, this hypothesis needs to be further investigated and validated through subsequent trials to advance the current develop ment in phage therapy.

Bacterial strains and growth conditions
The clinical strains used in this study were obtained from the Second Affiliated Hospital of Guilin Medical College and stored at −80°C in Luria-Bertani (LB) medium and solid medium (Qingdao Haibo Biotechnology Co.)

Isolation and purification of phage
The phage was isolated from the lake water of Guilin Medical College (Lingui District, Guangxi, China) and named SauPS-28.Isolation: 100 mL of the lake water was centri fuged at 10,000 ×g for 10 min at 4°C, and the precipitate was discarded.The supernatant was filtered using a 0.22-µm membrane (Millibol, Massachusetts, USA) and de-bacterized, followed by mixing with 100 mL of the LB broth and inoculation with MRSA at the logarithmic growth stage (OD600 about 0.6).Then, 1 mL of the bacterial broth was incubated at 37°C under 180 rpm shaking for 8 h, followed by centrifugation at 10,000 ×g for 10 min at 4°C and discarding of the precipitate.The supernatant was filtered through a 0.22-µm membrane to obtain the phage stock solution.Phage purification was performed using the DAP method.Briefly, the phage SauPS-28 stock solution series was diluted to 10 −8 , mixed with 100 µL dilution with 100 µL of the MRSA bacterial solution at each dilution gradient, and incubated at 37°C for 10 min.Then, semi-solid agar (warmed to 56°C) was added, mixed well, and poured onto the surface of the LB plates.After incubation of the plates at 37°C for approximately 13 h, the phage spots were enumerated on the plate.A sterile gun tip was used to pick clearer and smoother individual phage spots, which were then immersed in 1 mL of Tris-SM buffer, eluted with gentle shaking, and serially diluted for secondary screening.After four screenings, the phage spots of uniform size and typical characteristics were obtained.

Phage multiplication, concentration, and preservation
In accordance with the method of Gavric et al. (38), albeit with slight modifications, a single phage spot was inoculated with 100 mL of a logarithmic growth-phase MRSA solution, incubated at 37°C under 180 rpm shaking for 8 h, followed by centrifugation at 10,000 ×g at 4°C for 10 min, and the discarding of the precipitate.The supernatant was then filtered through a 0.22-µm filter membrane to obtain the phage culture.DNase I and RNase A were added to a final concentration of 1 µg/mL to the culture and left to stand at 37°C for 10 min.After which, sterile NaCl solution was used to make a final concentration of 1 mol/L, and the mixture was shaken well to mix on an ice bath for 1 h, followed by centrifugation at 8,000 ×g for 30 min at 4°C, addition of polyethylene glycol (PEG) 8000 to the supernatant to prepare a final concentration of 0.1 mol/L, gentle shaking to completely dissolve the PEG, and leaving overnight at 4°C.The next day, the mixture was centrifuged at 8,000 ×g for 30 min at 4°C, the supernatant was discarded, and the precipitate was resuspended with an appropriate amount of Tris-SM buffer to obtain a concentrated suspension of phage SauPS-28, which was then stored at 4°C.This suspension was used as the virulent species, freeze-dried, and stored at −80°C until further use.

Host-range determination
The host range of SauPS-28 was determined using the DAP method (39).A total of 22 MRSA strains were cultured to the logarithmic growth phase (OD600 approximately 0.6), and the phage solution was serially diluted.Then, 100 µL of the bacterial solution was mixed with different titers of the phage and added to semi-solid agar at 56°C, mixed, and the surface of LB solid-agar plates was covered in an incubator at 37°C and incubated for 12 h.The formation of phage spots indicated that SauPS-28 possessed lytic activities against the strain.

Transmission electron microscopy
A 150-mesh copper mesh with a carbon support film was used as a carrier mesh and irradiated under a UV lamp for 30 min.Then, 20 µL of a bacillus peptide was left to coat the copper mesh for 5 min and the filter paper was blotted dry.Also, 10 µL of the bacillus peptide was mixed with 10 µL of SauPS-28 and dropped onto the surface of the copper mesh and left for 20 min, after which the filter paper was blotted dry.Next, 20 µL of 2% (wt/vol) phosphotungstic acid was added to the surface for 60 s and the filter paper strips were blotted.The mesh was then placed in an airtight space and dried thoroughly.The Hitachi HT7700 TEM 80 kV was used for observation.

Multiplicity of infection
MOI is the ratio of PFU to colony-forming units (CFU) at the onset of infection.The MOI affects the efficiency and yield of phage proliferation.Phage SauPS-28 was mixed with the MRSA bacterial solution at various MOIs (0.001, 0.01, 0.1, 1, 10, and 100) and incubated at 37°C/180 rpm for 8 hours.DAP method was used to determine the phage titers.The ideal infection multiplicity is thought to be the one that produces the offspring phage with the highest titer.The experiment was repeated thrice under the same conditions and the results are expressed as mean ± SD.

Growth curve
The protocol of Huang et al. was employed, albeit with some minor modifications (53).The phage solution was mixed with the MRSA bacterial solution (1 × 10 8 CFU/mL; logarithmic growth stage) at an MOI of 0.01, and incubated at 37°C for 10 min, followed by centrifugation at 4°C and 12,000 ×g for 1 min.The supernatant containing free phage was discarded.Next, 10 mL of the LB liquid medium was resuspended and precipitated, followed by incubation at 37°C under 180 rpm.From this, 1 mL of the sample was removed every 10 min and the whole volume was made up of an equal volume of LB medium.The samples were then centrifuged at 4°C under 12,000 ×g for 1 min, the supernatant was removed, and DAP method was used to determine the phage titer of the samples at each stage.Next, a one-step growth curve of phage SauPS-28 was plotted to measure the latency and burst size.The experiment was repeated thrice and the mean was taken.Data were expressed as the mean and SD.

Acid-base stability
Different buffer pairs (such as sodium dihydrogen carbonate-disodium bicarbonate, citrate-sodium citrate, sodium hydroxide-potassium hydroxide, and sodium carbo nate-bicarbonate) were adjusted to obtain buffers with pH values 1-14.The SauPS-28 suspensions were mixed with buffers of different pH values, respectively, and incubated at 37°C for 1 h.The phage titers were determined using the DAP method.The experiment was repeated thrice, and the mean and SD values were plotted.

Thermal stability
The SauPS-28 suspensions were dispensed in centrifuge tubes and incubated at 4, 25, 37, 50, 60, and 70°C in a water bath for 0 min, 15 min, 30 min, 1 h, 2 h, 6 h, and 24 h, respectively, for the DAP determination of phage titers.The experiment was repeated thrice and plotted as the mean and SD.

Genome extraction and restriction digest mapping
SauPS-28 genome extraction was performed in strict accordance with the procedure of the Universal Phage Genomic DNA Extraction Kit (Item No. KG005-1, Nojing, Guangdong, China).An appropriate amount of SauPS-28 genome, restriction endonuclease EcoR I was digested at 37°C for 4 h and then separated by 0.7% agarose gel electrophoresis.

Phage proteome analysis
Concentrated SauPS-28 suspension (300 µL) was sonicated for 2 min at 360 W with a 10-s working interval.The protein concentration of the phage SauPS-28 was determined using the BCA method.Then, 34 µg of the phage protein was mixed with the loading buffer, boiled for 10 min, and subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation.The BIO-RAD Molecular Imager ChemiDoc XRS + Imaging System was employed for imaging.

Genome annotation and phylogenetic tree construction
Shanghai Heyuan Biotechnology Co., Ltd. was commissioned to complete the whole genome sequencing of phage SauPS-28 using the Illumina sequencing technology.NCBI BLASTn (http://blast.ncbi.nlm.nlm.nih.gov/) was applied for sequence identity matching and the online tool RAST (http://rast.nmpdr.org)was used for the functional annotation of ORFs.The sequences of 10 staphylococcal phage genomes with high sequence identity to SauPS-28 were downloaded, and the ClustalW comparison algorithm, maximum likelihood, based on MEGA-X software was used for sequence alignment, while a phylogenetic tree was constructed with Bootstrap repeated testing 1000 times to participate in the phylogenetic tree construction.GenBank accession numbers of the genomic sequences were as follows: B_UFSM1 (MW650841.1);B_UFSM3 (MW627293.1);

FIG 1
FIG 1 Bacterial plaque and TEM observation.(A) Double-layer agar assay of phage SauPS-28 with the host bacteria.(B) TEM observation of phage SauPS-28 with a positive polyhedral head structure, about 40 nm in diameter; showing an elongated tail of approximately 190 nm length and an overall length of approximately 230 nm.Scale bar = 100 nm.

FIG 2
FIG 2The growth curve of phage SauPS-28.The latency period of the phage is approximately 30 min, the lysis period is approximately 40 min, and the burst amount is approximately 25 PFU/cell.The experiment was repeated thrice, and the error line shows the mean ± SD.

FIG 4 FIG 3
FIG4 Thermal stability of phage SauPS-28.Phage SauPS-28 was incubated at 4°C, 37°C for 24 h and the activity was basically stable; at 50°C for 15 min, the titer of phage SauPS-28 decreased continuously and it became completely inactivated at 6 h; at 60°C or 70°C, phage SauPS-28 was completely inactive.The experiment was repeated thrice, and the error line shows the mean ± SD.

FIG 5
FIG5 The restriction digest profile of phage SauPS-28.Restriction endonuclease EcoR I was digested for 4 h, and multiple bands were separated using 0.7% agarose gel electrophoresis, indicating that SauPS-28 is a linear double-stranded DNA.

FIG 6
FIG 6 Separation of phage SauPS-28 structural proteins using SDS-PAGE gel.Phage SauPS-28 was treated with PEG8000, quantified using BCA, and denatured at 100°C, followed by separation on SDS-PAGE and staining with Coomassie Brilliant Blue-R250.The molecular weight of the ORFs corresponding to their functional annotation results was, in descending order, phage tail tape measure protein, terminase, phage major capsid protein, phage portal protein, and the hypothetical protein.The molecular masses of the proteins are marked on the far left panel.

FIG 7
FIG 7 Circos plot of the SauPS-28 genome.The outermost ring displays the distribution of different types of genes on the genome, with clockwise arrows representing genes in the positive direction in the genome and the counterclockwise table genomes representing genes in the negative direction.The red bars indicate the regions of the genome with higher than average GC content, while the dark blue bars indicate the regions of the genome with lower than average GC content.The yellow bars indicate the regions of the genome with positive GC skew, while the sky-blue bars indicate the regions of the genome with negative GC skew.

FIG 8
FIG8 Phylogenetic tree of the whole genome sequences of SauPS-28 phage and its related phages.The genomic sequences of these phages were compared based on the ClustalW comparison algorithm, ML, and a phylogenetic tree with 1000 bootstrap repeats using MEGA-X software.SauPS-28 is marked in red.

TABLE 1
The host-range spectrum of the phage SauPS-

28 a MRSA (No.) b Resistance Lysis ability Provider Isolation time
a

TABLE 3
Open reading frame (ORF) analysis of the phage SauPS-28 genome (Continued on next page)

TABLE 3
Open reading frame (ORF) analysis of the phage SauPS-28 genome (Continued)